Tum variants: immunogenic variants obtained by mutagen treatment of tumor cells

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59 Ford-Hutchinson, A. W. (1984)J. Allergy Clin. ImmunoL (Suppl.) 74, 437-440 60 Dahlen, S., Hansson, G., Hedgvist, P., Biorck, T., Granstrom, E. and Dahlen, B~ (1983) Proc. NatlAcad. Sci. USA 80, 1712-1716 61 Greaves, M. W. (1983)Br. J. DermatoL 109, 115-118 62 Sharon, P. and Stenson, W. F. (1983) Gastroenterol. 84, 1306 63 Rae, S. A., Davidson, E. M. and Smith, M. J. H. (1982) Lancet ii, 1122-1123 64 Cronwen, O., Walport, M. J., Morris, H., Taylor, G. W., Hodson, M. E., Batten, J. and Kay, A. B. (1981) Lancet ii, 164-165 65 Goetzl, E. J., Payan, D. G. and Goldman, D. W. (1984)J. Clin. ImmunoL 4, 79-84 66 Stenmark, K. R., James, S. L., Voelkel, N. F., Toews, W. H., Reeves, J. T. and Murphy, R. C. (1983) Ni Engl. J. Med. 309, 77-80 67 Goodman, M. G. and Weigle, W. O. (1980)J. ImmunoL 125, 593-600 68 Bailey, J. M., Bryant, R. W., Low, C. E., Pupilla, M. B. and Vanderhoek, J. Y. (1982) Cell. Immunol. 67, 112-120 68a Webb, D. R., Nowowiejski, I., Healy, C. and Rogers, T. J. (1982) Biochem. Biophys. Res. Commun. 104, 1617-1622 69 Rola-Pleszezynski, M., Borgeat, P. and Sirois, P. (1982) Biochem. Biophys. Res. Commun. 108, 1531-1537 70 Guttery, J. E., Tilden, A., Herron, D. K., Gallagher, P., Baker, S. R. and Ades, A. W. (1984)J. Clin. Lab. ImmunoL 13, 151-153 71 Payan, D. G. and Goetzl, E. J. (1983)J. ImmunoL 131, 551-553 72 Afluru, D. and Goodwin, J. S. (1984)Ji Clin. Invest. 74, 1444-1450 73 Gualde, N., Afluru, D., Goodwin, J. S. (1985)J. ImmunoL 134, 1125-1129 74 Payan, D. G., Missirian-Bastian, A. and Goetzl, E.J. (1984) Proc. Natl Acad. Sci. USA 81, 3501-3505 75 Gualde, N., Rabinovitch, H., Fredon, M. and Rigaud, M. (1982)Eur. J, lmmunoL 12, 773-777 75a Rola-Pleszezynski, M. and Sirois, P. (1983) in Leukotrienes and other Lipoxygenase Products(Piper, P. J. ed.) pp. 234-240, John Wiley & Sons 76 Rola-Pleszezynski, M. (1985),]: Immunol. 135, 1357-1360

77 Radoux, V., Gagnon, L., Pouliot, C., Corey, E. J. and RolaPleszczynski, M. (1984) Fed. Proc. 43, 1806 78 Rola-Pleszczynski, M. and Lemaire, I. Immunology in press 79 Dinarello, C. A., Bishai, I,, Rosenwasser, L.J. and Coceani, F. (1984) Int. J. ImmunopharmacoL 6, 43-50 80 Aldigier, J. C., Gualde, N., Mexmain, S., Chable-Rabinovitch, H., Ratinand, M. H. and Rigand, M. (1984) ProstaglandinsLeukotrienesMed. 13, 99-107 81 Myers, M.J., Ades, E. W., Jackson, W. T. and Petersen, B. M. (1984) J. Clin. Lab. Immunol. 15, 205-209 82 Goodwin, J. S., Gualde, N., Aldigier, J., Rigaud, M. and Vanderhoek, J. Y. (1984) ProstaglandinsLeukotrienesMed. 13, 109-112 83 Svenson, M., Bisgaard, H. and Bendtzen, K. (1984)Allergy 39,481-484 84 Johnson, H. M. and Torres, B. A. (1984)J. Immunol. 132, 413-416 84a Rola-Pleszczynski, M. and Lemaire, I. ProstaglandinsLeukotrienesMed. in press 85 Rola-Pleszczynski, M., Gagnon, L. and Sirois, P. (1983) Biochem. Biophys. Res. Commun. 113, 531-533 86 Rola-Pleszczynski, M., Gagnon, L., Rudzinska, M., Borgeat, P. and Sirois, P. (1984) ProstaglandinsLeukotrienesMed. 13, 113-117 87 Gagnon, L., Sirois, P. and Rola-Pleszczynski, M. (1984) Fed. Proc. 43, 1989 88 Mogbel, R., Sass-Kuhn, S. P., Goetzl, E.J. and Kay, A. B. (1983) Clin. Exp. Immunol. 52, 519-527 89 Ramstedt, U., Serhan, C. N., Lundberg, U., WigzeU, H. and Samuelsson, B. (1984) Proc. NatlAcad. Sci USA 81, 6914--6918 90 Seaman, W. E. (1983)J. Immunol. 131, 2953 91 Vanderhoek, J. Y., Ekborg, S. and Bailey, J. M. (1984)J. Allergy Clin. Immunol. 74, 412-417 92 Rola-Pleszczynski, M., Gagnon, L. and Sirois, P. (1984) in lcosanoidsand Cancer(Thaler-Dao, H., Crastes de Paulet, A. and Paoletti, R., eds), pp. 235-242, Raven Press 93 Hadden, J. W. and Coffey, R. G. (1982) ImmunoL Today 3, 299-304 94 Wess, J. A. and Archer, D. L. (1984) Int. J. Immunopharmavol. 6, 27-34

T u m - variants: immunogenic variants obtained by mutagen treatment of tumor cells Thierry Boon Mutagen treatment of mouse tumor cells produces at high frequency stable tumor cell variants that are rejected by syngeneic mice. As Thierry Boon discusses here, these 'turn-' variants express new suoCace antigens that can be recognized by cytolytic T lymphocytes. Tum- variants derived from spontaneous mouse tumors for which no immunogenicity had hitherto been demonstrated induce an immune protection against the parental tumor. Finally, he poses some interesting questions for future investigations. Over the past few years, it has become increasingly clear that, by treating mouse tumor cell cultures with mutagenic compounds, it is possible to obtain, at high frequency, tumor cell variants expressing new surface antigens. These variants elicit a rejection response in the syngeneic host 1. They have been named ' t u r n - ' because, unlike the 'turn +' cell from which they are derived, they fail to form progressive tumors. Here we review the main features of the turn- variants. Production of variants with increased immunogenicity W h e n clonal mouse tumor cell lines are exposed in vitro to the potent mutagen N-methyl-N'-nitro-N-nitrosoguanidine ( M N N G ) , the surviving cell population contains variants that are unable to form progressive tumors in normal adult syngeneic animals (Fig. 1). Such turn

Ludwig Institute for Cancer Research, 74 avenue Hippocrate, UCL 74.59, B-1200 Brussels, Belgium and Cellular Genetics Unit, Catholic University of Louvain, Brussels, Belgium.

variants have been obtained from many different mouse tumor cell lines, including teratocarcinoma 2, Lewis lung carcinoma 3, mastocytoma P8154, several spontaneous leukemias 5 and an adenoacanthoma 6. With a dose of M N N G allowing for approximately 0.1% survival of the initial cells, the frequency of turn- variants among the survivors usually ranges from 1 to 20 %. Repeated mutagenic treatments increase the frequency of variants. For instance, the frequency obtained with P815 was equal to 1, 20 and 95% after 1, 3 and 8 exposures to M N N G respectively 7. A large number of variants have been obtained that retain the t u m - phenotype during several months of continuous culture. For most of these variants, no change in morphology or growth rate was observed in vitro, and no gross karyotype alterations were noticed 24. Recently, the proteins of several variants derived from mastocytoma P815 were analysed in two-dimensional gel electrophoresis. From the thousand or more spots that could be clearly distinguished, at most one or two differences were .observed between turn + and turn- cells (K. Willard, ,personal communication). (~ 1985,ElsevierSciencePublishersB.V.,Amsterdam 0167- 4919/85/$02.00

308 mouse tumor

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Immunology Today, vol. 6, No. 10, 1985 turn- clones

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i ,oo+ i Several lines of evidence indicate that the failure of turn- variants to form progressive tumors is the consequence of an immune rejection response. First, turnvariants do form progressive tumors in mice that have been immunosuppressed by a sublethal dose of g a m m a radiation and also in nude mice 2-4.6. Moreover, for P815, which produces ascites, it is possible to follow the fate of turn + or t u m - cells injected in the peritoneal cavity by tapping the cavity every few days and counting the living tumor cells with an agar colony test. The results indicate that the turn- cells multiply exponentially during the first ten days. Around day 12-15 the tumor cells are eliminated completely in a few days and a large influx of lymphocytes and macrophages into the peritoneal cavity is observed 4. Finally, mice that have rejected a turnvariant are endowed with a radioresistant immune memory enabling them to reject a challenge of the same variant even when they receive immunosuppressive irradiation concurrently 2-4. This immune memory can be transferred adoptively with immune spleen T cells 2. W h e n mice are immunized against a turn- variant, they usually present a higher degree of resistance against a challenge with the same variant than against any other turn- variant derived from the same tumor cell line 3'a. This can be explained by assuming that most turnvariants have acquired a new transplantation antigen that is specific for each variant. As shown below, the existence of these 'turn- antigens' has been confirmed in vitro. Mutagenic compounds other than M N N G generate turn- variants in vitro. The triazenylimidazole derivative D T I C was shown by Bonmassar and his associates to generate t u m - variants in vitro in the presence of liver microsomes 9. They had indeed shown as early as 1970 that D T I C had a similar effect in vivo 1°. Ethyl methane sulfonate and azacytidine have also been used with success by Kerbel, Frost and their associates TM. These authors observed that, for several tumors, clones that appeared t u m - on a first screening formed tumors in a subsequent test. This initial instability of many turnvariants is not contradictory to our observation that most turn- variants that prove to be stable for one month continue to be so on long-term culture. Recently, Zbar and his associates showed that turn variants can be obtained at high frequency by treating a guinea pig fibrosarcoma with M N N G 12. This is the first time that the t u m - phenomenon has been observed outside the mouse species. It is of particular interest because, unlike the mouse genome, the guinea pig

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genome carries very few endogenous retroviral sequences. The involvement of retroviral proteins in the production of t u m - antigens is, therefore, less plausible than it may have appeared. Relation of turn - a n t i g e n s to T S T A Tumors induced by polycyclic hydrocarbons like methylcholanthrene usually express strong tumorspecific transplantation antigens (TSTA) 13-j5. The relation of these T S T A to the tumoral transformation process has been a long-sta~lding question that has not yet been solved despite the recent developments regarding oncogenes. Since most chemical carcinogens are proven mutagens, and since mutagens produce turn - antigens at very high frequencies, the possibility arises that the T S T A 'observed on most methylcholanthrene-induced tumors result from an effect of the carcinogen that is concomitant with, but independent of, that causing the tumoral transformation. Another very interesting system of tumors that express T S T A , which may have some relation with the t u m antigens, are the tumors induced by ultraviolet radiation. Kripke and her associates have shown that these tumors carry strong transplantation antigens provoking rejection by normal micC 6. The tumors, nevertheless, evolve progressively in the original UV-irradiated animals because of the induction of suppressor cells that appear to be directed against UV-induced tumors as a group ~7'1a. This tolerance to UV-induced tumors can be induced by grafting UV-irradiated skin on to normal mice 19. A recent report indicates that U V radiation can be used to obtain turn - variants from Lewis lung carcinoma 2°. Analysis o f t u m - antigens w i t h cytolytic T cells Unfortunately, it has been impossible up to now to obtain antibodies directed against turn - antigens. This is not surprising, considering the scarcity of specific antibodies obtained against T S T A of methylcholanthreneinduced tumors and against minor histocompatibility antigens. However, with some turn - systems it has been possible to observe a strong cytolytic T lymphocyte (CTL) response directed against turn- antigens 21'22. W e summarize here the results obtained with P815 turn variants since they have been the subject of the most extensive study with C T L . W h e n spleen cells from syngeneic mice that have rejected a variant are stimulated in vitro with the same variant, very active C T L are generated. In most

Immunology Today, vol. 6, No. 10, 1985

instances, these C T L show a definite specificity for the immunizing variant even though a significant lytic activity is observed against the turn ÷ cells and other turn variants 21. A systematic analysis of the C T L activities generated in response to more than 20 variants has led to the following conclusions~: 1. Approximately two-thirds of the turn- variants generate a variant-specific response, confirming the evidence obtained in vivo for new turn antigens. All gradations are seen, from variants that induce a cytolytic activity that is twice as high on themselves as on turn + target cells, to variants where this specificity factor is larger than ten. The variants that do not carry a turn - antigen detectable with C T L are nonetheless strongly rejected in vivo. 2. No t u r n - antigen has been found twice among more than 15 variants expressing these antigens. Moreover, no cross-reactive lysis involving any pair of turn variants has been observed, above that which can be accounted for by the T S T A already present on the original turn ÷ cells. Thus, the repertoire of the t u m - antigens is very likely to exceed 50 and may prove to be considerably larger. In this respect the turn - antigens are similar to the methylcholanthreneinduced TSTA. 3. No new antigen inducing a specific C T L response is observed on mutagen-treated cells that have remained turn + . 4. The C T L activity directed against most turnantigens appears to be H-2 restricted on the basis of inhibition with anti-H-2 antibodies 23. It should be noted that for some tumors strong C T L activities are observed with most turn - variants whereas for other tumors no C T L activity is observed against any turn- variants, even though the variants of the latter tumors are rejected as vigorously as those of the former. Such a lack of correlation between C T L activity and rejection response has been reported for other systems, such as the male-specific antigen H - Y 24'2s. The results obtained in vitro with immune spleen cells have been confirmed and extended by the clonal analysis of CTL. When stimulated in limiting dilution conditions in the presence ofIL-2, spleen cells from mice immunized with P815 turn - variants yield C T L clones at a frequency of 10-4-10 -3 (Ref. 26). With spleen cells that have already been stimulated in mass culture this frequency rises to 10 1. C T L clones that show a strict specificity for the immunizing turn- variant are obtained (Fig. 2). Others are directed against a T S T A of P815 (turn ÷ antigen); they lyse all P815 cells, whether turn ÷ or turn , but not syngeneic tumor L1210. M a n y C T L clones can be expanded and maintained in culture for several months without losing their activity and specificity. By using C T L clones directed against turn - antigens of P815, it has been possible to dissect these antigens into several components that can be lost independently of each other 27. Upon incubation of some turn- variants in the presence of the appropriate anti-turn- C T L clone, stable secondary variants can be obtained that appear to have lost the turn- antigen recognized by this CTL. In some instances, these antigen-loss variants have retained another antigenic determinant that is specific for the

309

original turn- variant. Other C T L clones can then be obtained that are directed against this second turnantigenic determinant. Thus, two components of the tuna- antigen can be selected independently of each other. When both are removed, the cells regain their ability to form tumors in normal micC 7. When an occasional injection of t u m - cells produces a progressive tumor, these tumor cells have also usually lost the turn antigen or at least one of its components 28(Fig. 2). C T L clones have also allowed the distinction of four different antigenic components in the T S T A present on the turn + P815 cells. Here also, antigen-loss variants are relevant to an interesting phenomenon encountered in vivo. Sometimes an intraperitoneal inoculum of P815 is almost completely eliminated by a rejection response, but a small number of residual tumor cells persists and remains stationary in the peritoneal cavity for a period that can exceed several weeks. This phenomenon has also been observed with mouse lymphoma L5178Y and has been referred to as the 'tumor dormant state' 29. Eventually the P815 tumor cells which have escaped rejection proliferate to form a progressive lethal tumor. These escaping cells have always lost one or two components of the T S T A of P8153°. A similar type of study, performed with UV-induced tumors which usually regress in normal mice, has indicated that there are also occasional 'progressor' tumors which have lost an antigenic componenC 1. Some of these progressors have also acquired an increased resistance against macrophage-mediated lysis 32. This has not been observed with P815.

Unsolved questions T u r n - variants are obtained at frequencies that are several orders of magnitude higher than those at which metabolic mutants are obtained after similar mutagen treatment. Epigenetic mechanisms involving regulatory molecules or reversible DNA modification such as methylation could, therefore, be involved. However, the stability of the turn - variants raises the possibility that the expression of new antigens couldbe caused by changes in DNA sequences. This in turn implies that either the genomic domain that determines the expression of turnantigens is enormous (more than 1000 genes) or that it is hypermutable or hyper-rearrangeable. tumor escape

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Immunology Today, vol. 6, No. 10, 1985

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A major step in our understanding of this problem would be to find out whether the large number of different turn - antigens constitutes a family of related molecules coded by a family of genes like the immunoglobulins or the major histocompatibility class I and class II molecules. O r are the tuna- antigens carried by an array of completely unrelated molecules? Due to the lack of antibodies, the biochemical isolation and characterization of t u m - antigens has not been achieved. Perhaps the cloning of the relevant genes will prove easier. This could be attempted by gene transfection since the expression of turn - antigens is dominant: somatic hybrids obtained by fusing one turn ÷ and one t u m - cell express the parental turn- antigen 3~. However, it first will be necessary to devise proper methods to detect transfectants with C T L . Protection conferred by t u m - variants against the o r i g i n a l t u m o r cells By rejecting a living inoculum of turn- variant ceils, mice acquire a certain degree of resistance against a challenge with the original turn + clone. This protection is weak: it is usually not effective against challenges that exceed a few times the minimum tumorigenic dose. However, it is long-term and specific for the original tumor 5,a. This protective effect was to be expected for turn: variants obtained from slightly immunogenic tumors like Lewis lung carcinoma. For P815, it is already more interesting because no protection can be obtained with irradiated turn ÷ cells, even though this tumor carries T S T A . But for teratocarcinoma cell line PCC4 the protection observed is truly remarkable as this tumor does not show any immunogenicity. Not only do irradiated PCC4 cells fail to immunize, but even when living cells are injected and form a subcutaneous tumor that is surgically removed after three weeks, no subsequent protection is observed. Nevertheless, PCC4-derived turn- variants confer a significant protection a. Thus, by immunizing with turn- variants one obtains a rejection response directed against TSTA. They behave like haptens in the sense that they do not elicit a rejection response on their own but are targets for a response elicited b y their association with another antigenic molecule. For most weakly immunogenic or non-immunogenic tumors, a protective immunization can be obtained with living t u m - cells, but not with irradiated turn - or turn ÷ cells. This is probably largely due to the fact that turnvariants provide a way of maintaining a high dose of immunogen for ten or more days during which the turn cells proliferate before their rejection. However, the results mentioned above regarding teratocarcinoma suggest that this is not the whole story since there a large dose of living turn ÷ cells fails to immunize. Also, one P815 t u m - variant confers protective immunity even when irradiated cells are used for immunization. The presence of a new turn antigen m a y therefore play a direct role in facilitating an immune response to a T S T A of the tum ÷ cell. Such an 'associative recognition' was suggested by Mitchison several years ago 34'35. Kobayashi and others obtained similar effects b y infecting t u m o r cells with viruses in order to generate new surface antigens ~6.

Presence of T S T A on spontaneous mouse tumors In contrast to the rodent tumors, obtained with oncogenic viruses, chemical carcinogens and with U V radiation, which are usually highly immunogenic, spontaneous tumors appear incapable of eliciting any rejection response 37'3a. Since turn- variants appeared to provide an efficient way of eliciting an immune response against very weak TSTA, it was tempting to examine whether their use could result in detection of T S T A on spontaneous tumors. Turn variants have been derived from two spontaneous leukemias obtained by Hewitt in C B A / H t mice. Some of these variants provide a significant protection against parental tumor cells that have never been adapted to culture 5. Also, mice immunized with turn - variants produce C T L directed specifically against the parental tumor. These results, which were obtained in conditions aimed at minimizing artefactual antigenicity, suggest that spontaneous tumors also carry weak T S T A . The only reservation is that the spontaneous tumors used for this work had been transplanted a large number of times. The possibility of acquisition of T S T A during these transplantations cannot be excluded. The turn - variants still provide us with more questions than answers. W h a t is the molecular nature of turn antigens? By what mechanism can mutagen induce new transplantation antigens at such high frequency? W h a t is the mechanism of rejection of these variants? Is it possible to obtain antigenic variants by treating human tumor cells with mutagens? And, will it be possible to improve the protective immunization obtained with t u m - variants up to a level where it may be of some therapeutic value? Much additional research will be requircd to provide the answers but progress appears possible. [~ References 1 Boon, T. (1983)Adv. CancerRes. 39, 121-151 2 Boon, T. and Kellermann, O. (1977) Proe. Natl Acad. Sd. USA 74, 272-275 3 Van Pel, A., Georlette, M. and Boon, T. (1979) Proc. NatlAcad. Sci. USA 76, 5282-5285 4 Uyttenhove, C., Van Snick, J. and Boon, T. (1980)J. Exp. Med. 152, 1175-1183 5 Van Pel, A., Vessiere, F. and Boon, T. (1983)J. Exp. Med. 157, 1992-2001 6 Frost, P., Kerbel, R., Bauer, E., Tartamella-Biondo, R. and Cefalu, W. (1983) CancerRes. 43, 125-132 7 Marchand, M., Casper, P. and Boon, T. (1983) Eur. J. Cancer Clin. Oncol. 19, 1529-1537 8 Boon, T. and Van Pel, A. (1978) Proc. NatlAead. Sd. uSA 75, 1519-1523 9 Contessa, A, R., Bonmassar, A., Giampietri, A., Circolo, A., Goldin, A. and Fioretti, M. C. (1981) CancerRes. 41, 2476-2482 I0 Bonmassar, E., Bonmassar, A., Vadlamudi, S. and Goldin, A. (1970) Proc. Natl Acad. Sci. USA 66, 1089-1095 11 Frost, P., Liteplo, R. G., Donaghue, T. P. and Kerben, R. S. (1984) J. Exp. Med. 159, 1491-1501 12 Zbar, B., Sukumar, S., Tanio, Y., Terata, N. and Hovis, J. (1984) CancerRes. 44, 5079-5085 13 Gross, L. (1943) CancerRes. 3, 326-333 14 Prehn, R. and Main, J. (1957)J. Natl CancerInst. 18, 769 778 15 Klein, G., SjSrgren, H., Klein, E. and Hellstr6m, K. E. (1960) Cancer Res. 20, 1561-1572 16 Kripke, M. (1974)J. Nat. Cancerlnst. 53, 1333-1336 17 Fisher, M. S. and Kripke, M. (1978)J. ImmunoL 121, 1139-1144 18 Spellman, C. and Daynes, R. (1977) Transplantation 24, 120-126 19 Palaszynski, E. and Kripke, M. (1983) Transplantation 36, 465-467 20 Peppoloni, S., Herberman, R. and Gorelik, E. (1984) Proceedingsof the 75th annual meetingof the American Associationfor CancerResearch, 25, 281 21 Boon, T., Van Snick, J., Van Pel, A., Uyttenhove, C. and Marchand, M. (1980)J. Exp. Med. 152, 1184-1193

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22 Vessiere, F., Georlette, M., Warnier, G., Leclerc,J.-G., Van Pel, A. and Boon, T. (1982)Eur.j . CancerClin. OrwoL 18, 867-874 23 Van Snick,J., Maryanski,J., Van Pel, A., Parmiani, G. and Boon, T. (1982), Eur. J. ImmunoL 12, 905-908 24 Hurme~ M., Hetherington, C. M., Chandler, P. R. and Simpson, E. (1978).]. Exp. Meal. 147, 758-767 25 Hurme, M., Chandler, P. R., Hetherington, C. M. and Simpson, E. (1978)J. Exp. Med 147, 768-775 26 Maryanski, J., Van Snick, J., Gerottini,J.-C. and Boon, T. (1982) Eur. a( Immunol. 12, 401-406 27 Maryanski,J. and Boon, T. (1982) Eur. J. Immunol. 12, 406-412 28 Maryanski,J., Marchand, M., Uyttenhove,C. and Boon, T. (1983)Int. J. Cancer31, 119-123 29 Weinhold, K., Miller, D. and Wheelock, E. (1979)dr. Exp. Med. 149, 745-757

30 Uyttenhove, C., Maryanski,J. and Boon, T. (1983)a( Exp. Med. 157, 1040-1052 31 Urban, J., Burton, R., Holland, M., Kripke, M. and Schreiber, H. (1982)J. Exp. Med. 155, 557-573 32 Urban, J. and Schreiber, H. (1983)J. Exp. Med. 157, 642-656 33 Maryanski, J., Szpirer, J., Szpirer, C. and Boon, T. (1983) Somatic Cell Gewaics9, 345-357 34 Mitchison, N. A. (1970) TranspL Pro¢. 2, 92-96 35 Lake, P. and Mitchison, N. A. (1976) Symp. Quant. Biol. 41,589-595 36 Kobayashi, H. (1982) in ImmunologicalAspects of Cancer Therapeutics(E. Mihich ed.) Wiley, New York 37 Hewitt, H. B., Blake, E. R. and Watder, A. (1976) Brit. J. Cancer33, 241-259 38 Middle, J. G. and Embleton, M. J. (198I) J. Natt Cancer Inst. 67, 637-643

) It was fitting that Paul Ehrlich's portrait was chosen in 1971 to grace the medal struck on the occasion of the First International Congress of Immunology. M a n y great scientists had contributed importantly to the founding and advancement of immunology during its first 90 years, but no one had so influenced the field as this man. It was he who first showed how to obtain high titre antisera, who introduced quantitative methods into immunology, and who defined the antigen-antibody interaction in stereochemical terms. It was Ehrlich also who helped to clarify the mechanisms involved in immune hemolysis, who introduced the notion of cell surface receptors, and who performed the first experiments to show the immunological relationship between mother and fetus and newborn. Finally, it was he who advanced the first selective theory of antibody formation, who proposed immunoregulatory mechanisms in connection with his famous precept horror autotoxicus, and who anticipated the modern interest in idiotypes and antiidiotypes with his studies and speculations on anti-antibodies. Here was a man who was at once a superb experimentalist and an imaginative theoretician. But Ehrlieh's interests were not restricted to immunology. While still a student, his experiments with aniline dyes and their application to the staining of cells and tissues caused a minor revolution in histology and histopathology. His application of these staining techniques to blood smears founded the science of hematology. Later, he made significant contributions to cancer research, and his investigations leading to the discovery of trypan-red therapy for trypanosomiasis and of salvarsan (compound 606) therapy for syphilis founded the field of chemotherapy. Is it possible in a single biography to do full justice to so broadly-ranging a scien-

Paul Ehrlich: Scientist for life by Ernst Biiumler (English translation by Grant Edwards), Holmes & Meier, 1984. $39.50 (xvi + 288 pages) I S B N 0 8419 0837 0

tist, and at the same time not slight the man himself? Sir Henry Dale, who worked with Ehrlich in 1904, was 'disposed to think that an attempt to write Ehrlich's biography at full l e n g t h . . , would encounter particular difficulties'. The details of a scientific biography 'would be very difficult to make interesting, or even intelligible, beyond the circle of a relatively small n u m b e r of experts'. O n the other hand, the attempt to lighten the scientific burden for the general reader by discussing Ehrlieh's persona 'might easily, by contrast, take on the aspects of caricature'. Unfortunately, Sir Henry was correct; no one has yet succeeded in capturing both the man and his science. At least six biographies of Paul Ehrlieh have appeared prior to the present volume. The two earliest ones - by his former secretary Martha Marquardt ('Paul Ehrlich', New York, Henry Schuman, 1951) and by his former student, A. Lazarus, ('Paul Ehrlich', Vienna, Rikola Verlag, 1922) form the

cornerstone of all later efforts, but these may be coloured by deep affection for the Master. In general, the other biographies offer only the occasional new fact or scientific evaluation, and are principally concerned with the chemotherapeutic aspects of Ehrlich's work. These are: G. Venzmer, 'Paul Ehrlich, Leben und Wirken', Stuttgart, M u n d u s Verlag, 1948; H. Loewe, 'Paul Ehrlich, Sch6pfer der Chemotherapie', Stuttgart, Wissenschaftliche Verlagsges., 1950; W. Greiling, 'Ira Banne der Medizin: Paul Ehrlich', Dfisseldorf, Econ Verlag, 1954; and H. Satter, 'Paul Ehrlich, Begrfinder der Chemotherapie', MfincherL V e d a g R. Oldenbourg, 1963. Ernst B~umler, writer of the present biography of Paul Ehrlich, is Director of Community Relations for the pharmaceutical firm of Hoechst, a position which brings to this new effort both advantages and disadvantages. Since Hoechst was long associated with Paul Ehrlich - first in a joint venture with Emil von Behring to market diphtheria antitoxin in the 1890's, and later as Ehrlich's collaborator in the development and exploitation of his chemotherapeutic agents - B~mmler has been able to add to the Ehrlich story new excerpts from his voluminous correspondence with Hoechst personnel. In addition, B/iumler obtained access to some previously unrecorded correspondence between Ehrlich and his dear American friend, Christian A. Herter, all of which adds to our knowledge of the man and the scientist. But counterbalancing this, divided Hoechst loyalties may have coloured a portion of Bgumler's story. One of the saddest events of Ehrlich's life was his falling out with his old friend yon Behring. After yon Behring's discovery (with Kitasato) of diphtheria antitoxin in 1890, Ehrlich showed how to obtain it in useful high titres, and how to measure it. Together they signed an agreement with Hoechst in 1894 to market ~ i s great therapeutic advance, from whicI~ both should have derived great financial benefit. Sometime later, Ehrlich was induced to forego his portion

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