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ALFRED NISONOFF January 26, 1 923-March 12, 2001 BY LISA A. STEINER, KATHERINE L. KNIGHT AND ]. D O NALD CAPRA AEFRED NISONOFF, WHO DIED on March ~ 2, 2001, was a ma- jor contributor to many basic aspects of immunology throughout his career. In aciclition to funciamental work that helpecl to define the nature of antibodies en cl the genes encocling them, he was an astute critic with penetrating analytical skills. His monograph The Antibody Molecule stancis as the clefinitive reference work on the subject to 1975, the time of its publication. NisonofEs parents immigrated to the New York area from Hungary en cl Russia as teenagers. Al was born in Corona on January 26, 1923. When he was about two years oicI, his parents movecl into a working-cIass, largely immigrant com- munity in South River, New Jersey, to join other family mem- bers. They operates! a kosher butcher shop en c! grocery store. Al's parents had little formal education and his ini- tial exposure to books en cl reacting was in school, where his exceptional intelligence was soon recognized. At age 6 he founcl himself in third gracle, en cl by 15 he hacl gracluatecl from high school. One of the few students in his school to go to college, Al receiver! a state scholarship en c! enrollee! at Rutgers, which was within hitchhiking distance en cl al- lowocl him to live at home. He became interested in chem- 161

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62 B I O G RA P H I C A L EMOIRS istry when a high-school frienc! gave him access to his home laboratory, en cl cleciclecl to major in this fielcI. It also seemed to offer opportunities for practical future employment. Upon graduation from Rutgers in 1942 at age 19, Al set off in a Moclel A Forcl to take up a job with the U.S. Rubber Company in Detroit. It was the first time he hacl been more than 50 miles from home. He later recallec! being toic! by an upper management person that he shouIcl be prowl because U.S. Rubber clicl not orclinarily hire Jews. He was probably not surpriser! to hear this, because it was generally accepted at this time that many chemical companies wouIcl not hire Jews (see Dan A. Oren, Joining the Club: A History of Jews en c! Yale, p. 357, footnote 28. New Haven: Yale University Press, 1985~. Although Al was assigned to a fairly routine task, testing various latex compounds for their ability to adhere to the nylon coral required to strengthen airplane tires, he soon macle a critical observation that changecl the procluction of these tires so important for the war effort. One clay while walking through the plant, he stopped to watch the con- struction of self-sealing gasoline tanks macle from rubber and strengthened with nylon cord dipped in a water-based latex adhesive. Combining the keen power of observation en cl imagination that was to characterize his future research, Al aciaptec! this process to the problem of adhering nylon coral to rubber tires so U.S. Rubber was launchecl into making nylon-beltecl tires. Describing this practical cliscov- ery many years later, Al with typical self-deprecation said it was "primitive stuff . . . a mincIless sort of thing." Another significant event of the time in Detroit was that Al met Sarah (Sally) Weiseman at a Jewish community center. They corresponclecl through the war years en cl were married im- mecliately after his discharge from the Navy. By 1943, with the war raging, Al became anxious to join

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ALFRED NISONOFF 163 the armec! forces. Even though he hac! an occupational cle- ferment, he enlistecl as a midshipman in the Navy. He served until the end of the war, missing the invasion of Okinawa only because his ship clevelopec! an engine problem. He hacl not given much thought to the future, but a college friend whom he met while passing through San Diego toIcl him about the G.I. Bill, en c! he cleciclec! to pursue graduate work in chemistry. He was clischargecl from the Navy in July 1946 en cl entered graduate school at Johns Hopkins Uni- versity in September, receiving his M.A. in 1948 en c! Ph.D. in 1951. His research, supervised by Frederick W. Barnes, fir., was on the enzymatic mechanism of transamination. Following graduate school en c! on the strength of his previous success at U. S. Rubber, Al joined a branch of the same company in Naugatuck, Connecticut. After two years, however, he cleciclec! to return to work relater! to biochem- istry en cl took a position with David Pressman's group at the Roswell Park Memorial Institute in Buffalo, beginning work that set the direction for much of his research in the remainder of his career. In the early 1940s David Pressman, then working in Linus PauTing's group, carrier! out an extensive series of experi- ments exploring the specificity of antibodies clirectecl against haptenic determinants. These studies introclucecl the tech- nique of quantitative hapten inhibition, an important ex- tension of the experimental approach pioneered by Karl Lancisteiner. Throughout his career Nisonoff appliecl quan- titative approaches, often with anti-hapten antibodies, to a number of problems in immunology. With Pressman, Nisonoff explorecl the heterogeneity in the bincling of antibodies with haptens en c! introclucec! means of estimating this het- erogeneity quantitatively. In an important paper from this period he clemonstratecl that the two combining sites on a single antibody molecule have the same specificity. This

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64 B I O G RA P H I C A L EMOIRS result, which confirmed! earlier experiments by Lancisteiner, Felix Haurowitz, en cl Herman Eisen using precipitin meth- ocis, was important in that it renclerecl unlikely that specific antibody sites would simply be generated by folding around an antigen template, as hacl been specified in the "instruc- tion" theories of antibody formation, in most cletail by Pauling. Towarc! the ens! of his stay at Roswell Park, Nisonoff initiated experiments on the enzymatic cleavage of rabbit antibodies, which contributed importantly to the growing unclerstancling of their structure. Rodney Porter hac! shown that two active univalent fragments, now known as Fab, couIcl be proclucecl from each antibody molecule by digestion with papain. Because papain is always user! in the presence of a mercaptan, Nisonoff originally proposal that two steps, pro- teolysis en cl clisulficle cleavage, were neeclecl to generate the active univalent fragments. Accordingly, he repeated Porter's experiment with a different enzyme, pepsin, which floes not require activation by a mercaptan. Although the initial premise was incorrect, as Nisonoff himself later pointer! out, the experiment lecl to an even more interesting con- clusion. Disulficle bond cleavage is not required to produce the active univalent antibody fragments after papain cleav- age, but it is required to produce univalent fragments after limitecl digestion with pepsin. The explanation is that pa- pain cleaves on the amino-terminal sicle of the single clisul- fide bridge connecting the two heavy chains in rabbit IgG, whereas pepsin cleaves on the carboxyI-terminal sicle of the same bond, generating a single bivaTent fragment, F(ab')2. Reduction of the inter-heavy chain bridge in the bivalent fragment yields univalent Fab' fragments. Nisonoff's studies provided critical insights into the na- ture of the fragments proclucecl by digestion with papain en cl their disposition in the intact antibody molecule. His experiments with pepsin digestion implies! that the two frag-

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ALFRED NISONOFF 165 meets containing the active site (Fate or Fab') are locater! on the same sicle of the molecule, away from the Fc frag- ment. The cleliberations by Porter's group that lecl to the formulation of the polypepticle chain structure of IgG were summarized by Julian FIeischman in a "citation classic" re- view of the work. As FIeischman recallecI, Porter initially favorer! the then popular cigar-shape mocle! in which the two active fragments are clisposecl on either sicle of a cen- tral Fc. However, this moclel was not easily reconcilecl with Nisonoff's results with pepsin digestion en c! was therefore abanclonecl in favor of the now familiar four-chain moclel in which the two Fab fragments are on one sicle of the molecule. Nisonoff's work also ciarifiec! the nature of chro- matographic fractions I en cl II obtained by Porter after pa- pain digestion of rabbit antibodies. The similar yielcl ini- tially fount! for fractions I en c! II was fortuitous, the result of charge heterogeneity in the antibody population en cl the choice of column conditions. In fact, the more negatively charger! antibody molecules were fount! to contain two Fab fragments of type I en cl the more positively charged two Fab fragments of type II. The F(ab')2 fragment proclucec! by pepsin retains the bivalence of the original antibody molecule en cl therefore the ability to precipitate or agglutinate antigen. However, it lacks the Fc fragment en c! will not bins! to cells expressing Fc receptors, eliminating much unclesirecl "non-specific" antibody bincling. The next logical step, taken by Nisonoff just as he was moving from Roswell Park to a position as associate professor of microbiology at the University of Illi- nois, Urbana, was to show that the univalent Fab' fragments generated by successive pepsin digestion and reduction could be recombined into the bivalent F(ab')2 fragment by oxicia- tion, allowing the creation of bivalent antibodies of mixecl specificity. Such hybric! antibodies have hac! many practical

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66 B I O G RA P H I C A L EMOIRS uses, for example, bringing a pharmacological agent into contact with a particular cell type. Throughout his career Nisonoff retained an interest in the three-climensional structure of antibodies. As early as the late 1950s this lecl to collaboration with Cecil Hall en cl Henry Slater at the Massachusetts Institute of Technology in an attempt to visualize the antibody molecule by elec- tron microscopy. Although resolution was insufficient to discern the shape, the ciata proviclecl a reasonable estimate of the size of the molecule. Methods of X-ray crystallogra- phy began to be applied to proteins in the 1960s. Recogniz- ing the importance of applying these techniques to anti- boclies, Nisonoff soon succeeclec! in obtaining crystals of Fab fragments clerivecl from human IgG myeloma proteins. Preliminary structural work was carried out in colIabora- tion with Roberto Poijak's group, later more detailed stud- ies carried out by PoIjak en cl others lecl to a cletailecl uncler- stancling of the structure of the Fab fragment, inclucling localization of the active site, the basic features of the Ig foIcI, en cl the orientation of V en cl C domains. In 1966 Nisonoff movecl from Urbana to the University of Illinois College of Medicine in Chicago, where in 1969 he assumed the chair of the Department of Biological Chem- istry. In Chicago he continual a fruitful collaboration with Shelclon Dray, relating structural features of rabbit antibocI- ies to genetic variations known as allotypy. In work using Nisonoff's characteristic quantitative approach, they showocl that the population of IgG molecules in a rabbit heterozy- gous for allotype consists only of molecules displaying one or the other allotypic determinant, but not both. Coming on the heels of the proposal of the four-chain mocle! for IgG, this finding suggested that the IgG molecule is sym- metrical (i.e., with two iclentical heavy chains en cl two iclen- tical light chains).

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ALFRED NISONOFF 167 Thinking that quantitative immunochemical methods wouIcl also be useful for investigating icliotypy (the unique antigenic specificity possessed by incliviclual antibody mol- ecuTes), Nisonoff embarked! on a series of studies that lair! the groundwork for the wiclespreacl use of icliotypes as ge- netic markers. IncleecI, studies of icliotypy were to occupy him for the rest of his career. He cirew on his experience with an tiboclies clirectecl against haptenic cle termin ants to aciciress such questions as the relationship of the icliotypic site of an antibody molecule to the antigen-bincling site. In other studies Nisonoff aciciressecl such questions as the size of the repertoire of antibocly-bincling sites en cl the relation- sh~p of the ~ct~otyp~c site of an antibody molecule to the antigen-binding site. For example, an important insight pro- viclecl by Nisonoff, as well as others, was the recognition that some icliotypic specificities can be sharer! by different inclivicluals, so-callecl public icliotypes, en cl that these are orobabIv encoded bv Caroline penes. The relationship be- . . ~ .. . .. . .. ~ ... . . - J ~ - -- -J a- - a- --I - - 1- tween icliotypes en c! genes encocling antibody V regions pro- viclecl means for tracking clonal lines of B-lymphocytes. In the 1960s evidence began to accumulate indicating that a single germline gene conic! not encocle both the variable en cl constant regions of an Ig heavy or light chain. One of the critical finclings was proviclecl by Nisonoff, who in collaboration with Hugh Fudenher~ shower! that IgG en c! lglVl myeloma proteins ootalnect trom tne same patient had iclentical icliotypes, en cl therefore iclentical V regions, as was later confirmed! by sequence analysis. Because the C regions of the gamma en cl mu chains must be encoclecl by distinct genes, the V and C segments had to be specified by separate genetic units. He showocl that antibodies clirectecl against icliotypic cle- terminants couIcl compete with hapten in bincling to anti- bocly-combining regions. Nisonoff en c! l. DonaTc! Capra in a T ~ ~ ~ , ~ - --a-- - -- -- ----- --a --- ~ . . ~ ~ . ~

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68 B I O G RA P H I C A L EMOIRS collaboration that extenclec! over a number of years shower! that icliotypes couIcl be clefinecl structurally en cl that icliotypic differences between different strains of mice reflect genetic variability. With Paul GottTieb, Nisonoff proviclec! evidence that both heavy en cl light chains in an immunogIobulin molecule are required for expression of the icliotype. Nisonoff's work en c! that of others on icliotypy is summa- rizecl in his presiclential aciciress for the American Associa- tion of Immunologists meeting of 1991. A major occupation en c! preoccupation of the later Chi- cago years was the writing, together with John Hopper en cl Susan Spring, of the monograph The Antibody Molecule (1975), a monumental annotates! work providing a schol- arly en cl comprehensive review of what we now call the B- cell receptor. Choosing to review the fielcl at this time was a reflection of Nisonoff's astute insight, for it proviclec! a cle- finitive summary of the protein phase of molecular immu- nology en cl set the stage for the genetic era that was soon to follow. A clecacle later Nisonoff wrote an introductory text of molecular immunology that showocl not only his superb command of the subject but also his skill in present- ing complex material. His extensive knowlecige en c! clear, rigorous thinking macle him an excellent teacher both in the laboratory en cl in the classroom. In 1975 Nisonoff mover! to the Rosenstie! Research Center at Brancleis University, where he helpecl to bring younger colleagues to a group whose focus was research in immu- nology. His own work continued to be centered on idiotypy. Interestingly, he was recruited to Brandeis by Harlyn Halvorson, then director of the Rosenstiel center, whose father, H. Orin Halvorson, had brought Nisonoff to the Microbiology Department at the University of Illinois, Ur- bana, at the outset of his academic career. Nisonoff's well-deserved reputation for critical scientific

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ALFRED NISONOFF 169 insight en c! sounc! judgment meant that he was much sought after to serve on review panels, advisory committees, en cl eclitorial boards. In what may be a record, he served three terms, once as chair, on the allergy en c! immunology stucly section of the National Institutes of Health. He was presi- clent of the American Association of Immunologists in 1990- 91. Even after giving up his research program in 1996, Nisonoff continual as an Divisor to NIH en cl playocl a ma- . . Or role In preparing a comprehensive report on current knowlecige en c! future directions in immunology (Report of the NIAID Task Force on Immunology, National Institutes of Health, 1998~. Retirement was not easy for him, as he misses! the give en c! take of the lab, but he remainec! as sharp as ever en cl was always available for criticism en cl clis- cussion with former colleagues. One of Nisonoff's outstanding qualities was intellectual honesty. Always direct and outspoken, he disliked pretense in any form en cl was himself without any pretension. He saw right to the heart of any question en c! brooked! no fuzziness of thought. The high stanciarcis he set for himself were a moclel for colleagues, as well as for the many stu- clents he mentorecI. He pair! little attention to the external trappings of fame, his or anyone else's. He was interested in science for its own sake en cl was forever in pursuit of the rigorous experiment that wouic! provicle an unambiguous answer to a meaningful question. Nisonoff's characteristic modesty is epitomized in the format of his curriculum vitae in which his honors are bur- ied in a section captioned "Other Data." He received the Mecial of the Pasteur Institute in 1971, was a foreign corre- sponclent of the Beigian Academy of Medicine in 1977, a fellow of the American Academy of Arts en cl Sciences in 1982, en cl became a member of the National Academy of Science in 1984.

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170 B I O G RA P H I C A L EMOIRS As a young person Nisonoff hac! little exposure to the visual arts or to music, but he came to love classical music passionately en cl was a regular at Boston Symphony con- certs. He lover! the works of many twentieth-century com- posers but was generally not enamored of the newly com- missionecl works that are often incluclecl in these concerts. He was mover! to express this view in a letter sent to the Boston Symphony just a few months before his cleath. Why, he askocI, were new compositions so rarely playocl again after their premiere? If they are worth hearing once, he reasoned, surely they are worth hearing twice. As far as is known, the question remained unanswered. Nisonoff's sense of fun en c! goof! humor were legencI- ary. In aciclition to music, he lovecl to play tennis en cl clicl so regularly. He was invariably goocl for a lively discussion of the current political scene. He hac! a strong lifelong sense of social justice en cl always took the sicle of the unclerclog. He quietly set about cloing what he couIcl to make his cor- ner of the woric! a better place. After retirement from Brancleis he coached kicis in math en cl recent immigrants in English, and he was planning to take a course on teach- ing English as a seconc! language. Nisonoff was as honest in his human relationships as he was in his science. To colleagues en cl students, as well as family en c! friencis, he was loyal en c! committed. Although he was clivorcecl from Sally in 1978, he continual to care for her on a ciaily basis. He hacl a close relationship with his chiTciren, Don en c! Lincia, en c! was a clevotec! grandfather. He was the best friend of his younger sister, Lorraine. The last clecacle of his life was immensely enriched by his friencI- ship with Patricia Carella, who sharer! his love of music en c! travel. Al Nisonoff was one of a small number of investigators whose accomplishments span the classical en c! moclern eras

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ALFRED NISONOFF 171 in immunology. His work proviclec! critical insights into the molecular nature of the antibody molecule en cl the genetic basis for antibody diversity. He approached important bio- Togical questions with the rigor of the chemist. His uncler- stancling of this complex fielcl was as broacl as it was creep. His publications s ten cl as a moclel of clear thinking en cl ~ vlslon.

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172 B I O G RA P H I C A L EMOIRS SELECTED BIBLIOGRAPHY 1958 With D. Pressman. Heterogeneity and average combining constants of antibody from individual rabbits. 7. Immunol. 80:417. With D. Pressman. Heterogeneity of antibody sites in their relative combining affinities for structurally related haptens. 7. Immunol. 81:126. 1959 With M. H. Winkler and D. Pressman. The similar specificity of the combining sites of an individual antibody molecule. 7. Immunol. 82:201. With C. E. Hall and H. S. Slayter. Electron microscopic observa- tions of rabbit antibodies. 7. Biochem. Biophys. Cytol. 6:407. 1960 With F. C. Wissler and D. L. Woernley. Properties of univalent frag- ments of rabbit antibody isolated by specific adsorption. Arch. Biochem. Biophys. 88:241. With F. C. Wissler, L. N. Lipman, and D. L. Woernley. Separation of univalent fragments from the bivalent rabbit antibody molecule by reduction of disulfide bonds. Arch. Biochem. Biophys. 89:230. With F. C. Wissler and L. N. Lipman. Properties of the major com- ponent of a peptic digest of rabbit antibody. Science 132:1770. 1961 With G. Markus and F. C. Wissler. Separation of univalent frag- ments of rabbit antibody by reduction of a single, labile disulfide bond. Nature 189:293. With M. M Rivers. Recombination of a mixture of univalent anti- body fragments of different specificity. Arch. Biochem. Biophys. 93:460. 1962 With J. L. Palmer and W. J. Mandy. Heterogeneity of rabbit anti- body and its subunits. Proc. Natl. A cad. Sci. U. S. A. 48:49.

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ALFRED NISONOFF 1963 173 With S. Dray. Contribution of allelic genes Ab4 and A be to forma- tion of rabbit 7S 7-globulins. Proc. Soc. Exp. Biol. Med. 113:20. 1964 With A. M. Gilman and S. Dray. Symmetrical distribution of genetic markers in individual rabbit 7-globulin molecules. Immunochem- istryl:109. 1965 With R. Hong. Relative [abilities of the two types of interchain di- sulf~de bonds of rabbit ~G-immunoglobulin. 7. Biol. Chem. 240:3883. 1968 With G. Rossi. Crystallization of fragment Fab of human IgG my- eloma proteins. Biochem. Biophys. Res. Comm. 31:914. With H. P. Avey, R. J. Poljak, and G. Rossi. Crystallographic data for the Fab fragment of a human myeloma immunoglobulin. Nature 220:1248. With S. Zappacosta and W. J. Mandy. Mechanism of cleavage of rabbit IgG in two stages by soluble papain and reducing agent. 7. Immunol. 100:1268. 1969 With H. Daugharty, J. E. Hopper, and A. B. MacDonald. Quantita- tive investigations of idiotypic antibodies. I. Analysis of precipi- tating antibody populations. 7. Exp. Med. 130:1047. 1970 With A. B. MacDonald. Quantitative investigations of idiotypic anti- bodies. III. Persistence and variations of idiotypic specificities during the course of immunization. 7. Exp. Med. 131:583. With B. W. Brient. Quantitative investigations of idiotypic antibod- ies. IV. Inhibition by specific haptens of the reaction of anti- hapten antibody with its anti-idiotypic antibody. J. Exp. Med. 132:951. With A. C. Wang, S. K. Wilson, J. E. Hopper, and H. H. Fudenberg. Evidence for control of synthesis of the variable regions of the heavy chains of immunoglobulins G and M by the same gene. Proc. Natl. A cad. Sci. U. S. A. 66:337.

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174 B I O G RA P H I C A L 1972 EMOIRS With D. A. Hart, A.-L. Wang, and L. L. Pawlak. Suppression of idiotypic specificities in adult mice by administration of anti-idiotypic antibody. J. Exp. Med. 135:1293 With M. G. Kuettner and A.-L. Wang. Quantitative investigations of idiotypic antibodies. VI. Idiotypic specificity as a potential ge- netic marker for the variable regions of mouse immunoglobulin polypeptide chains. J. Exp. Med. 135:579. With R. J. Poljak, L. M. Amzel, H. P. Avey, and L. N. Becka. The structure of Fab' "New" at 6 A resolution. Nature New Biol. 235:137. 1973 With L. L. Pawlak, E. B. Mushinski, and M. Potter. Evidence for the linkage of the IGCH locus to a gene controlling the idiotypic specificity of anti-'azophenylarsonate antibodies in strain A mice. J Exp. Med. 137:22. 1974 With K. Eichmann and A. S. Tung. Linkage and rearrangement of genes encoding mouse immunoglobulin heavy chains. Nature 250:509. 1975 With J. D. Capra and A. S. Tung. Structural studies on induced antibodies with defined idiotypic specificities. I. The heavy chains of anti-p-azophenylarsonate antibodies from A/J mice bearing a cross-reactive idiotype. 7. Immunol. 114:1548. With J. E. Hopper and S. B. Spring. The Antibody Molecule. New York: Academic Press. 1977 With S.-T. Ju and A. Gray. Frequency of occurrence of idiotypes associated with anti-p-azophenylarsonate antibodies arising in mice immunologically suppressed with respect to a cross-reactive idiotype. 7. Exp. Med. 145:540. With J. A. Laskin, A. Gray, N. R. Klinman, and P. G. Gottlieb. Segre- gation at a locus determining an immunoglobulin genetic marker for the light chain variable region affects inheritance of expres- sion of an idiotype. Proc. Natl. A cad. Sci. U. S. A. 74:4600.

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ALFRED NISONOFF 1981 175 With A. R. Brown and E. Lamoyi. Relationship of idiotypes of the anti-~azophenylarsonate antibodies of A/J and BALB/c mice. 7. Immunol. 126: 1 268. 1984 Introduction to Molecular Immunology. Sunderland, Mass.: Sinauer Associates. 1991 American Association of Immunologists Presidential Address. Idiotypes: Concepts and applications. 7. Immunol. 147:2429.