From Proteins and Protein Models to Their Use in Immunology and Immunotherapy (2003)

The two leitmotifs of my scientific research were synthetic polymeric models of proteins and the structure of proteins as reflected by the three-dimensional conformation controlled by their amino acid sequence. This in turn led me to synthetic polypeptide antigens and to investigation of antibodies, ultimately resulting in the discovery of the determinant-specific genetic control of immune response and to the development of a therapeutic vaccine against the exacerbating-remitting form of multiple sclerosis. In protein chemistry I had two great teachers who became very close friends, Ephraim Katchalski-Katzir and Christian B. Anfinsen (Fig. 1), but in immunology I lacked the benefit of a great mentor, and so to some extent I was self-taught. Thus, I had to “figure out” many things myself. In my childhood I grew up in the courtyard of a textile factory, and I later intended to study fibers, mainly the new synthetic ones. However, I first started studying macromolecules when, as a Ph.D. student, in 1950 I joined the laboratory of Ephraim Katchalski at the Weizmann Institute of Science. Ephraim first synthesized polylysine in the early 1940s and was successfully exploring the use of polyamino acids as protein models. With his collaborators he prepared and studied many physical, chemical, and biological properties of several polymers of trifunctional amino acids such as polyarginine, polyaspartic acid, polyhistidine, and polyserine, as well as the polyamino acids polyproline and polyhydroxyproline (1.Katchalski E. Sela M. Adv. Protein Chem. 1958; 13: 243-492Crossref PubMed Scopus (158) Google Scholar, 2.Sela M. Katchalski E. Adv. Protein Chem. 1959; 14: 391-478Crossref PubMed Scopus (87) Google Scholar). Among the early syntheses was the one of poly-l-tyrosine, which was part of the subject of my Ph.D. thesis. My thesis research included also the synthesis of poly-3,5-diiodotyrosine and poly-p-amino-l-phenylalanine. Later on I was directly involved with the synthesis of polytryptophan and polycyclohexylalanine. Of special interest was the spectrophotometric titration of polytyrosine and of copolymers of tyrosine with positively or negatively charged amino acids; this showed the influence of the vicinal electrostatic field on the ease of ionization of the phenolic hydroxyl groups (2.Sela M. Katchalski E. Adv. Protein Chem. 1959; 14: 391-478Crossref PubMed Scopus (87) Google Scholar, 3.Sela M. Katchalski E. J. Am. Chem. Soc. 1956; 78: 3986-3989Crossref Scopus (14) Google Scholar). We were very touched when John Edsall included figures from this study in his book Biophysical Chemistry with Jeffreys Wyman. Actually Proteins, Peptides and Amino Acids as Dipolar Ions (Cohn and Edsall's book) served as a basis for intense seminars with both of the Katchalski-Katzir brothers, Ephraim and Aharon. The accent was on physicochemical properties of proteins, polyamino acids, and polyelectrolytes in general. Ephraim was a remarkable teacher—stimulating, inspiring, patient, and always friendly. Today I still cherish his friendship. For the preparation of poly-l-tyrosine, I remember that I tried 40 times to synthesize the monomer, N-carboxy-O-benzyloxycarbonyl-l-tyrosine anhydride, before I was successful. This taught me the need for perseverance and optimism in research. Of course, serendipity (defined as luck meeting the prepared mind) also helps. I mention this because much of the story that follows actually depended on the availability of this monomer (4.Katchalski E. Sela M. J. Am. Chem. Soc. 1953; 75: 5284-5289Crossref Scopus (50) Google Scholar). My first paper was on the titration of N-carboxy-α-amino acid anhydrides in nonaqueous solvents (5.Berger A. Sela M. Katchalski E. Anal. Chem. 1953; 25: 1554-1555Crossref Scopus (18) Google Scholar). These are the monomers in the synthesis of amino acid polymers, and the titration was useful for the determination of unreacted anhydride at any instant. My Ph.D. thesis was on the azo derivatives of some aromatic poly-α-amino acids (6.Sela M. Katchalski E. J. Am. Chem. Soc. 1955; 77: 3662-3663Crossref Scopus (3) Google Scholar). One could even diazotize poly-p-aminophenylalanine and couple it to polymers containing tyrosine, resulting in colored water-insoluble compounds. I shall describe later how the work with polyamino acids brought me into immunology. What I would like to mention here is that we could use as initiators of polymerization of N-carboxyamino acid anhydrides (the monomers from which polyamino acids were built) not only monofunctional small molecules but also macromolecules possessing several amino groups. If proteins were used as such polyvalent initiators, we ended up with polypeptidyl proteins, whereas when polylysine or polyornithine were used, we had for the first time multichain polyamino acids, which became so important for me later as synthetic antigens. Mark Stahmann also prepared such polypeptidyl proteins in Wisconsin. We summarized in Advances in Protein Chemistry in 1958 (1.Katchalski E. Sela M. Adv. Protein Chem. 1958; 13: 243-492Crossref PubMed Scopus (158) Google Scholar) and 1959 (2.Sela M. Katchalski E. Adv. Protein Chem. 1959; 14: 391-478Crossref PubMed Scopus (87) Google Scholar) the early studies on the synthesis and the chemical and biological properties of poly-α-amino acids. In 1955 I was back in Europe as a young scientist presenting the work on multichain polyamino acids at the International Congress of Chemistry in Zurich, and a week later, the work on spectrophotometric titration of polymers and copolymers of amino acids at the International Congress of Biochemistry in Bruxelles. This was a wonderful occasion to meet scientists whom until then I had known only by reputation and through their papers. Among the many I want to mention two, Hans Neurath and Bill Harrington, who later became close friends of mine. During the same trip I went to see Sir Charles Harington in London. Harington was the man who elucidated the structure of thyroxine and predicted that it is formed from two diiodotyrosine residues by oxidation rather than simple dismutation. I wanted to tell him that the availability of polytyrosine had permitted us (7.Sela M. Sarid S. Nature. 1956; 178: 540-541Crossref Scopus (11) Google Scholar) to find out that, following alkaline incubation of iodinated polytyrosine, the hydrolysate contained 2% thyroxine and 2% serine. We had, thus, proved the validity of his prediction. Later I shall discuss our work on linear and multichain polyamino acids as synthetic polypeptide antigens, but now I want to mention that ultimately we developed a therapeutic drug/vaccine against the exacerbating-remitting stage of multiple sclerosis, denoted copolymer 1, named glatiramer acetate and Copaxone by the industry. This drug, approved by the Food and Drug Administration in 1996 and used by tens of thousands of patients, is the first polymeric drug/vaccine in which the active ingredient is the polymer itself, in contrast to polymers used earlier in pharmacopeia for slow release of drugs or for wrapping them. As a result of studying the mechanism of polymerization of N-carboxyamino acid anhydrides with Arieh Berger, I was involved in an interesting story that I want to relate here while discussing polyamino acids. On my second visit to the National Institutes of Health (NIH) in 1960, Marshall Nirenberg came one day and asked me whether I had some poly-l-phenylalanine and whether I knew its solubility properties. I did not have the polymer in Bethesda, but I did ask Nirenberg why he was interested. Through these conversations I became one of the first to know about the breaking of the genetic code, UUU encoding Phe. Although I was somewhat skeptical of the story, I immediately looked for and found, hidden somewhere in an experimental section of a paper in the Journal of the American Chemical Society, that poly-l-phenylalanine was insoluble in all the solvents we had tested with the exception of a saturated solution of anhydrous hydrogen bromide in glacial acetic acid (8.Sela M. Berger A. J. Am. Chem. Soc. 1955; 77: 1893-1898Crossref Scopus (84) Google Scholar). Because on that very day I was preparing just such a solution (used to remove carbobenzoxy groups) in the laboratory, I gladly gave the reagent to Nirenberg and was touched and surprised when he acknowledged this in the classical paper that resulted in his receiving the Nobel Prize. The real point of the story lies elsewhere. Why did we try to use such a peculiar solvent? The truth of the matter is that years earlier, together with the late Arieh Berger in Rehovot, we were investigating the mechanism of polymerization leading to linear and multichain polyamino acids. One day I had two test tubes, one with polyphenylalanine and one with polycarbobenzoxylysine, stuck in a beaker on my desk. Arieh came to decarbobenzoxylate the lysine polymer, a reaction with hydrogen bromide in glacial acetic acid during which carbon dioxide is released. He took the wrong test tube away with him and returned, puzzled because the material had dissolved and he could not see any evolution of carbon dioxide. At once we realized the mistake and I noted in my laboratory book that, at long last, we had found a solvent for poly-l-phenylalanine. After 5 years of working with protein models, I felt ready to go abroad for a postdoctoral period to work with proteins. Thus, I arrived in 1956 in the laboratory of Chris Anfinsen at the NIH in Bethesda. The friendship between us resulted in prolonged stays in Bethesda in 1956–1957, 1960–1961, and 1973–1974. On his part, Chris came to Rehovot on sabbaticals on several occasions and was an active, extremely valuable member of the Board of Governors of the Weizmann Institute of Science, serving for many years as the Chairman of its Scientific Advisory Committee. His death in May 1995 was for me personally a deeply felt loss. Chris was a great friend, helpful to all those who surrounded him, full of charm and modesty, and actually a great romantic. From the NIH we sent out our first joint paper, which Chris was in a hurry to prepare for a Festschrift honoring Linderstrom-Lang. The topic was the selective splitting of protein chains by trypsin at arginine residues after lysine residues were reversibly blocked by carbobenzoxy groups. Oxidative opening of the four disulfide bridges of bovine pancreatic ribonuclease to permit its sequencing was possible only because tryptophan was absent in this protein. A more general method, which could be used also for a protein containing tryptophan, such as lysozyme, was reductive cleavage, followed by blocking of the sulfhydryl groups with iodoacetic acid (9.Sela M. White Jr., F.H. Anfinsen C.B. Biochim. Biophys. Acta. 1959; 31: 417-426Crossref PubMed Scopus (56) Google Scholar). I was fortunate to participate with Chris in these studies and left part of the reduced ribonuclease without blocking its sulfhydryl groups to see if it could reoxidize properly and whether the enzymatic activity would come back. The results of these experiments (10.Sela M. White Jr., F.H. Anfinsen C.B. Science. 1957; 125: 691-692Crossref PubMed Scopus (154) Google Scholar) showed a total recovery of the activity even though statistical considerations pointed to 105 various ways in which the four disulfide bridges could reform. Thus, we demonstrated that no additional information was needed for the correct, unique architecture of a protein molecule and that it is the genetic code dictating the amino acid sequence that is responsible for the conversion of the randomly coiled structure. It is for this most important observation that in 1972 Chris was awarded the Nobel Prize in chemistry, which he shared with Stanford Moore and William Stein, who established the amino acid sequence of ribonuclease. I learned a lot from Chris, a dedicated scientist, who had an incredible flair for attacking the right protein and the most elegant experiment to solve it, a flair matched only by his literary talent. During my visits to Bethesda, I tried in two ways to combine my previous experience with polyamino acids and research on enzymes. One was the use of copolymers of glutamic acid with aromatic amino acids to efficiently inhibit ribonuclease (11.Sela M. J. Biol. Chem. 1962; 237: 418-421Abstract Full Text PDF PubMed Google Scholar), lysozyme, or trypsin. The other series of studies had to do with poly-dl-alanylation of proteins. In contrast to poly-l-alanine and poly-d-alanine, poly-dl-alanine, which is a random copolymer of l-alanine and d-alanine, is well soluble in water and may serve as a solubilizer. Poly-dl-alanyl ribonuclease could be reduced and reoxidized properly. Poly-dl-alanylation renders gliadin water-soluble (12.Sela M. Lupu N. Yaron A. Berger A. Biochim. Biophys. Acta. 1962; 62: 594-596Crossref PubMed Scopus (4) Google Scholar) and converts myosin into a derivative soluble in distilled water and with all its ATPase activity preserved (13.Edelman I.S. Hoffer E. Bauminger S. Sela M. Arch. Biochem. Biophys. 1968; 123: 211-221Crossref PubMed Scopus (6) Google Scholar). On a lighter level, when I arrived in Bethesda everybody was writing the sequences of amino acids in straight lines; the disulfide bridges (at straight angles to the chain) occupied as much space as 10 residues. I found a huge hole puncher with which I made round pieces of paper, wrote the name of 1 residue on each piece of paper, and played with them until the half-cystines of a bridge were able to touch each other. This gave rise to the well known “swan” shape of ribonuclease, and it delights me to see that proteins are still often schematically presented like this. After returning from my first visit to the NIH (1956–1957), I embarked on a collaboration with Nathan Citri on two conformationally different states of penicillinase. The enzyme changes from an iodine-resistant to an iodine-sensitive state in urea or guanidine hydrochloride. The change in conformation was followed by optical rotatory measurements. This, in 1960, was my first of many papers published in the Journal of Biological Chemistry (14.Citri N. Garber N. Sela M. J. Biol. Chem. 1960; 235: 3454-3459Abstract Full Text PDF PubMed Google Scholar). During my second stay at the NIH (1960–1961), I continued studies on oxidation of reduced ribonuclease (15.Anfinsen C.B. Haber E. Sela M. White Jr., F.H. Proc. Natl. Acad. Sci. U. S. A. 1961; 47: 1309-1314Crossref PubMed Scopus (897) Google Scholar) and started investigating the enzymatic properties of poly-dl-alanyl derivatives of ribonuclease (16.Anfinsen C.B. Sela M. Cooke J.P. J. Biol. Chem. 1962; 237: 1825-1831Abstract Full Text PDF PubMed Google Scholar, 17.Wellner D. Silman J.I. Sela M. J. Biol. Chem. 1963; 238: 1324-1331Abstract Full Text PDF PubMed Google Scholar), trypsin, and chymotrypsin (18.Epstein C.J. Anfinsen C.B. Sela M. J. Biol. Chem. 1962; 237: 3458-3463Abstract Full Text PDF Google Scholar). Alanylated ribonuclease with as many as 4 or 5 alanine residues per chain kept its enzymatic activity and regenerated its full activity after reduction of its disulfide bridges and subsequent reoxidation (16.Anfinsen C.B. Sela M. Cooke J.P. J. Biol. Chem. 1962; 237: 1825-1831Abstract Full Text PDF PubMed Google Scholar). We later learned that alanylation affected the enzymatic activity toward RNA but not toward the low molecular weight substrates (17.Wellner D. Silman J.I. Sela M. J. Biol. Chem. 1963; 238: 1324-1331Abstract Full Text PDF PubMed Google Scholar). Poly-dl-alanyl trypsin, with an average polyalanine chain length of 6–9 residues, was resistant to autolysis at temperatures up to 38 °C and reacted normally with soybean and serum inhibitors. Poly-dl-alanyl chymotrypsin had similar stability and activity properties (18.Epstein C.J. Anfinsen C.B. Sela M. J. Biol. Chem. 1962; 237: 3458-3463Abstract Full Text PDF Google Scholar). Several years later, Roger Acher visited us from Paris and investigated poly-dl-alanyl Kunitz trypsin inhibitor (19.Acher R. Chauvet J. Arnon R. Sela M. Eur. J. Biochem. 1968; 3: 476-482Crossref PubMed Scopus (6) Google Scholar). Only when the alanine residues were attached exclusively to the α-amino group was the inhibitor active. Upon alanylation of all the amino groups, the derivative was virtually inactive, demonstrating the crucial role of ϵ-amino groups for its activity. While still in Bethesda, I checked the inhibition of ribonuclease by copolymers of glutamic acid and aromatic amino acids (11.Sela M. J. Biol. Chem. 1962; 237: 418-421Abstract Full Text PDF PubMed Google Scholar). Copolymers of tyrosine (or phenylalanine) and glutamic acid are much more efficient inhibitors of ribonuclease than polyaspartic acid, and in this case the attachment to the enzyme occurs not only through electrostatic interactions but also through short range urea-labile bonds. The digestion of ribonucleic acid by pancreatic ribonuclease was stopped completely, at pH 5.0, by relatively small amounts of the inhibitory copolymer. We tested these copolymers also for inhibition of lysozyme (20.Sela M. Steiner L.A. Biochemistry. 1963; 2: 416-421Crossref PubMed Scopus (24) Google Scholar) and trypsin (21.Rigby M. Sela M. Biochemistry. 1964; 3: 629-636Crossref PubMed Scopus (7) Google Scholar) with similar results. As my Ph.D. thesis was concerned with polytyrosine (a polymeric chain of phenols) and poly-p-aminophenylalanine (a polymeric chain of anilines), it was natural to produce polypeptidic azo dyes from them. I reasoned that these might serve as synthetic models for azoproteins, of which one rare example, provided by Landsteiner, was the attachment of haptens including peptides, via an azo bond, to proteins. Reading Landsteiner's book The Specificity of Serological Reactions, I came across the statement that gelatin probably is not because it no This led me to study the of the of gelatin by attachment of tyrosine do these the amino groups of the protein were used to the polymerization of the tyrosine monomer, as The of this study was the Ph.D. thesis of and we showed that without whereas more gelatin into a mainly to M. Arnon R. Biochem. J. 1960; 75: PubMed Scopus (50) Google Scholar). It was at this time that we first the of and and it from I was to describe the results on the in of gelatin its to Sir Charles Harington on the same visit in as it was in his laboratory that John some years earlier had tried to the of gelatin by it to It was several years later that we started with John and his on several of synthetic polypeptide antigens, to a of immunology. As a result of the studies on we that gelatin was not for the gelatin with multichain M. Scholar, M. Katchalski E. M. J. Am. Chem. Soc. 1956; 78: Scopus Google Scholar) as the for of tyrosine and glutamic acid and showed that the resulting denoted led to in experimental At that stage joined and we synthesized linear and multichain polyamino acids and tested them for on a was by the that the not papers that are part of a so we published it M. Arnon R. Biochim. Biophys. Acta. 1960; PubMed Scopus Google Scholar). The paper M. S. Arnon R. Biochem. J. 1962; PubMed Scopus Google Scholar) became a The availability of synthetic antigens permitted a of the molecular basis of S. Sela M. Biochem. J. 1963; PubMed Scopus Google Scholar, M. Adv. PubMed Scopus Google Scholar, M. Science. PubMed Scopus Google Scholar). We could a lot about the role of and shape as well as about the of those of the molecule crucial for As a matter of we learned that it was possible one was prepared to the to prepare synthetic leading to of any Although in most a had a molecular of at several and were by of an efficient immune The for a molecule to be on its chemical Although may be important in the of an is not a for we could prepare water-soluble amino acid copolymers of that were of acids were only when were in amounts and led to no was as was that of several other polymers such as linear and multichain polymers of in with polymeric chains of and The of all these antigens was that not only but also were if at though polymers exclusively of acids are we were able to more that are of the of The different of acids had me for many and I summarized the topic M. E. J. PubMed Scopus Google Scholar). of the studies on the molecular basis of were out before the crucial role of in immune response was Only later the of immunology or not In the early was a wonderful working on synthetic antigens because was working on the but later on it was as and to know that so many had in the synthetic to One of the most of our studies with synthetic antigens had to do with the conformation of the and of its We between and M. B. Biol. Google Scholar) and showed how the same may to the sequence attached to multichain or an by conformation the was to an In we could for the first by how to the polymer could into a shape a small polymer that was not B. A. Sela M. Eur. J. Biochem. PubMed Scopus Google Scholar). These studies led us directly to study proteins and to synthesize a in which a synthetic from lysozyme was attached to polyalanine R. E. Sela M. Anfinsen C.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The resulting reacted with lysozyme through the but the reaction was when the disulfide the was and the three-dimensional structure was In this it be that of proteins may still be the we were investigating might have contained a of against from the In later years we showed that a of amino acid residues from the protein of after with an the of that with the Arnon R. Sela M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). tens of to of proteins have prepared that may to with the protein. one remember that many similar have prepared that were not of The extent of on the that the be able to the conformation that it in the protein. This may be when the is not able to a or when it is long and a conformation different from the one of If the protein is more the of is even though be when a small is of a relatively conformation similar to the one it a protein. Thus, to the with A. S. Sela M. Biochemistry. Scopus Google Scholar). The to the is also very important to out is most antigens the study of the role of in the of antibodies, and The results led us to the that an is much more than an attached to an we did not know that the of by and was the for our results. However, we did state that the had a crucial role in the of the immune response toward an to the between and for might be the we of between two of I came back to this when together with of M. Adv. Protein Chem. PubMed Google Scholar). in for we to gelatin from by R. Sela M. Science. 1960; PubMed Scopus Google Scholar). In case D. S. Sela M. Biochim. Biophys. Acta. 1962; PubMed Scopus (11) Google Scholar), we on of to such low molecular weight antigens as lysozyme and a synthetic With Arnon we in preparing an of the by splitting the molecule with bromide rather than with Arnon R. Sela M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. Arnon R. Sela M. J. 125: PubMed Scopus (4) Google Scholar). Poly-dl-alanylation of because of its great permitted the total reduction of all the disulfide bridges the molecule without the and chain This for controlled leading to the of and chains and the recovery of both and activity Sela M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, Sela M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). It also the that no genetic information other than that in the amino acid sequence of the polypeptide chains is for the conformation of a protein molecule as as to Rehovot from laboratory, he the interest in the to its was able to the with the first at that a D. Sela M. J. Biol. PubMed Scopus Google Scholar). Of great interest to me was the observation we made with on the between an and the it M. E. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar,