In search of the eighth factor: a personal reminiscence
2003; Elsevier BV; Volume: 1; Issue: 3 Linguagem: Inglês
10.1046/j.1538-7836.2003.00163.x
ISSN1538-7933
Autores Tópico(s)Cancer-related gene regulation
Resumo‘As failures go, attempting to recall the past is like trying to grasp the meaning of existence. Both make one feel like a baby clutching at a basket ball: one's palms keep sliding off the sides.'Joseph Brodsky [1Brodsky J. Less Than One: Selected Essays. 1986.Google Scholar] ‘Frances, if this succeeds you are going to be rich and famous!’ Victor Hoffbrand remarked. ‘No’ she replied, ‘David Heath will be rich, Ted will be famous and I’ll be out of work'. and that is more or less what happened. This exchange took place in the hemophilia center laboratory at the Royal Free Hospital c. 1983, during the heroic period when Frances Rotblat and Don O'Brien were making one milligram of pure factor (F)VIII every 3 weeks to send to Genentech for protein sequencing. It was the time of the cloning race; a race in which four groups vied for the academic glory and commercial rewards of being the first to synthesize FVIII. In what follows, I will attempt to recall the build up to the race, its progress and its outcome. My introduction to the problem of FVIII was a messily practical one. As a senior house officer in Pathology at Royal Liverpool Infirmary in 1970, I had to thaw and draw up into large syringes a viscous yellow liquid called cryo-precipitate and then inject it into the veins of patients with hemophilia to stop them bleeding. A rule of thumb was to use about five bags for a joint bleed and hope for the best. I do not think we checked the dose or the response but I do remember one patient whose bleeding failed to stop. After several doses we checked for an inhibitor and sure enough Alan Smith, then as now the coagulation technician in the hematology laboratory, found one. This, of course, was bad news for the patient as we had no way of treating him after that, but it sparked my interest in the whole area of hemostasis. That interest was fanned into a flame by my good fortune in working under the direction of Arthur Bloom in Cardiff, Wales, where I went as a lecturer in the Haematology Department in 1971. Arthur had set up a pioneering hemophilia center and research unit with special interest in FVIII and von Willebrand factor (VWF). At that time, since the two activities always seemed to purify together, most researchers considered them to be aspects of the same molecule, although evidence was beginning to accumulate that they might be separable under some conditions. One morning in 1972, Arthur called Ian Peake, John Giddings and myself into his office to propose a new theory about the two activities. Beginning with a summary of relevant published work, he set out the idea that there was a large molecule under autosomal genetic control that had VWF activity and a smaller molecule under X chromosomal control that had the antihemophilic factor activity. These two were associated in plasma but could be separated by high salt buffers. This hypothesis tied together all the clinical, genetic and laboratory data about hemophilia A and von Willebrand's disease and recent work from Zimmerman and Owen in the USA. However it would take more than a decade to provide the absolute biochemical and genetic proof of this visionary concept. With hindsight the real difficulty was the extremely low concentration of FVIII in plasma, its tendency to break down into inactive fragments and hence the great difficulty in getting enough to see on a gel and begin biochemical studies as opposed to purely functional and immunological ones. In 1973, Arthur funded me to go to the ICTH meeting in Vienna where Stefan Magnusson announced the complete sequence of bovine prothrombin and Gary Nelsestuen explained the action of warfarin in blocking carboxylation of glutamate residues on prothrombin. That got me hooked. Finding the sequence of FVIII became my life's goal for the next 10 years. At the meeting I presented some data on the presence of FVIII antigen (measured as inhibitor neutralizing activity) and FVIII-related antigen (at that time the name given to VWF antigen) in fetal development. I can still remember the severe epinephrine rush produced by standing in front of an international audience for the first time. After 4 happy years in Cardiff, where I learned enough clinical and laboratory medicine to pass the two Royal College membership examinations, Arthur got me a job as a research assistant in Leon Hoyer's laboratory in Connecticut, USA. Lee had been developing the discovery by Ted Zimmerman that rabbit antibodies to FVIII/VWF could be made which bound the complex of the two activities. Lee showed that by immobilizing such antibodies on sepharose gel the whole FVIII/VWF complex could be captured from plasma and then FVIII activity on its own could be eluted with a buffer containing 0.24 mol L−1 calcium chloride, leaving the VWF antigen behind. I arrived in Connecticut on the first day of January 1976, the depths of New England winter. For the next 3 months I traveled to work and left work in the dark through streets with the snow piled up to 6 feet high on the sidewalks. It was very exciting both to be in America for the first time in my life and to be working full time on the FVIII problem. We gradually increased the size and loading of the columns. By developing a highly sensitive immunoradiometric assay for VWF antigen I could show that what eluted from the columns had up to 10 U mL−1 of FVIII activity but tiny or undetectable amounts of VWF antigen. This seemed to us to prove that they were separate entities. Furthermore, this free FVIII was highly sensitive to thrombin and could be bound to concanavalin A, proving that it was glycoprotein in nature. Because we had to use albumin in the buffers to prevent non-specific loss of protein on the columns and glassware it was impossible to determine the specific activity of this free FVIII. However when the time came to publish this work, we encountered stiff resistance from a reviewer chosen by the Journal of Clinical Investigation who evidently did not accept the two molecules hypothesis, and we had to send the paper to the Journal of Laboratory and Clinical Medicine[2Tuddenham E.G. Trabold N.C. Collins J.A. Hoyer L.W. The properties of factor VIII coagulant activity prepared by immunoadsorbent chromatography.J Lab Clin Med. 1979; 93: 40-53PubMed Google Scholar]. To this day, I have not succeeded in getting a paper into the Journal of Clinical Investigation. Returning to England after 2 years in the USA, I was appointed as a locum consultant in the Haemophilia Centre set up by Katharine Dormandy at the Royal Free Hospital Medical School in Hampstead, London. Katharine was then bravely fighting the terminal stages of cancer. After she took early retirement I applied for her post as Senior Lecturer and was appointed. The first task was to move the Center into its new and splendid purpose built facilities. Katharine had approached the father of one of her patients (Clive Knight) in order to raise the money for this initiative. After Katharine's death the center was named for her. Upstairs in the new but almost bare laboratories I started to build a team for a renewed assault on FVIII. First to join me was Frances Rotblat, then a trainee hematologist, for whom we obtained a small grant from the hospital trustees to set up an immunoradiometric assay for FVIII antigen. This depended on finding a patient inhibitor sample with a very high antiFVIII titer. One of our patients was a charming young man called Colin Campbell who had inhibitors to FVIII but that had fallen to quite a low titer, as he had not been treated for a year. He came in with a particularly bad knee bleed, which started when he was umpiring a cricket match. My colleague Elenor Goldman suggested we try a high dose of FVIII, which seemed to work, although no measurable level of activity appeared in his blood. A week later the inhibitor went up to a historically high level of 8000 BU mL−1. Colin agreed to let us plasmapherese him to obtain a large sample of what was to become a uniquely valuable reagent. Frances used purified immunoglobulin from this plasma to set up an assay for FVIII antigen based on the one developed by John Lazarchick in Lee Hoyer's laboratory. This assay gave us a precise, sensitive and, most importantly, specific way of measuring FVIII independent of its coagulant activity and was used for the rest of the project. Indeed we and many other laboratories have used this antibody for many years in a wide range of studies. By screening about a hundred patients for their FVIII activity and antigen we identified several with normal levels of antigen but low or absent activity, the so-called cross-reacting material (CRM) positive cases. This was not new but later study of the same cases showed them to have particularly interesting mutations. In parallel with this work I obtained a Medical Research Council grant to study the interaction of FVIII with FIX and platelets. This required purification of FIX from clinical concentrate using methods developed by the legendary laboratory of Earl Davie. Later attempts to publish this work met with extremely harsh and indeed rude criticism from a reviewer chosen by the Journal of Biological Chemistry. Instead of persevering and resubmitting to another journal, I let this work stay in the drawer. Others have subsequently published very similar results. This is a lesson not to be discouraged by negative reviews of what one believes to be sound work. However a very valuable spin off of this unpublished work was that when Alison Goodall came round to ask if anyone had interesting proteins she could use to raise monoclonal antibodies, we were able to present her with fairly pure FVIII/VWF complex and FIX. Alison had recently joined George Janossy's Immunology Department at the Royal Free Hospital, London, UK, and had set up Cesar Milstein's novel and technically demanding method for making mouse monoclonal antibodies. This was to prove a highly successful collaboration and gave us the key to unlock the final purification of FVIII. We obtained a grant from The Medical Research Council to fund making monoclonal antibodies to clotting factors which enabled us to hire Don O'Brien. Don had a 2.1 degree in Biochemistry from The City of London Polytechnic College and had been paying his rent by working in a hospital laboratory whilst looking for a suitable research post. He came to us with considerable practical skill, boundless energy and a wicked sense of humor. It was definitely an advantage that he did not know how many PhD projects in the past had foundered on the heretofore-impossible task of purifying FVIII. Later, his brother Fergal joined us. First we made several monoclonal antibodies to FIX, which were amongst the first to be directed against a human coagulation protein. One of these antibodies had extremely high affinity and when immobilized on sepharose enabled subtractive depletion from whole plasma to create artificial FIX deficient plasma for use in the one stage assay, thus replacing the use of plasma from severely affected patients with hemophilia B. This encouraged us to attempt to make monoclonal antibodies specific for FVIII and for VWF. For the latter we immunized with a size-separated fraction from commercial concentrate, a very crude immunogen by today's standards, and screened by a two stage-binding assay. Remarkably we obtained two antibodies that were highly specific for the functional binding site of VWF and later formed the basis of an immunologic assay that very closely paralleled the functional assay. However, for the purposes of FVIII purification it served very well to capture the whole complex, just like the rabbit antibodies I had used in Connecticut. At this point we were not much closer to making enough pure FVIII to immunize even a mouse. However developments in clinical concentrate production gave us a new angle on the problem. Alan Johnson in New York had been trying out novel polyelectrolyte resins which had the remarkable property of preferentially adsorbing FVIII compared to VWF and fibrinogen, the main protein contaminants of all FVIII concentrates up to that time. This caught the attention of David Heath, a pharmacologist and the director of a small company called Speywood based in Nottingham, that was making a concentrate of FVIII from pig blood. David Heath had bought the rights to make pig FVIII from Maws, the company who commercialized MacFarlane's animal FVIII concentrates. Bovine and porcine FVIII concentrates had been the therapeutic breakthrough of the 1950s at the Oxford Haemophilia center. Robert Gwyn Macfarlane, codiscoverer of the coagulation cascade, was unable to source enough human plasma for FVIII concentrate production and turned to the virtually unlimited source of blood to be had from slaughterhouses. The blood of pigs and cows has about five times as much FVIII per milliliter as human blood, according to activity assays. The concentrates made at Oxford and later by Maws were used in many desperate clinical situations and saved many hemophiliacs' lives but they had severe drawbacks. Pig and cow VWF aggregate human platelets in the circulation causing thrombocytopenia, they are highly immunogenic which practically limits their use to single episodes, the concentrates were highly insoluble and they also produced anaphylactic reactions. Clearly higher purity could alleviate the immediate problems although not the immunogenicity. David Heath's chief scientist Sarah Middleton, showed that polyelectrolyte purified porcine FVIII made using Alan Johnson's method was readily soluble, did not cause thrombocytopenia, had high specific activity and a lower incidence of allergic reaction on infusion. We started to use the new concentrate for patients with antibodies to human FVIII and achieved remarkable success in those patients whose antibodies had low cross-reactivity to pig FVIII. My colleague at the center, Peter Kernoff, was instrumental in introducing this advance to the clinic. We even found some patients who tolerated long-term use of the new porcine FVIII concentrate without developing resistance. One of those patients still uses porcine FVIII for home treatment. The UK National blood fractionation laboratory at Elstree became interested and produced a quantity of polyelectrolyte purified human FVIII for us to use in a small-scale clinical trial. This material was quite low in VWF and had fairly high specific activity, around 100 U mg−1 of protein. The hemophiliacs we treated showed a good response and the half-life of infused FVIII activity in their circulation was 10 h, similar to conventional FVIII complex. However the PE purified FVIII had a half-life of only 2.5 h when infused into a young man with severe von Willebrand disease. Furthermore he had no secondary rise in FVIII activity, as happens when such patients are infused with any source of VWF. This was the first in vivo demonstration that one role of VWF is to preserve FVIII in the circulation, as predicted by Arthur Bloom. Frances and I were mulling over these results and discussing how to use them in further purifying FVIII when it came to both of us simultaneously that there might be enough VWF in the PE purified concentrate for the FVIII to still be absorbed to a column of antibody to VWF bound to sepharose. But in this case because of the high ratio of FVIII to VWF much more FVIII would be first bound to, then eluted from the column with 0.24 mol L−1 calcium chloride buffer. We could hardly wait to try it out. Sure enough the PE purified FVIII absorbed completely to the antibody column and the eluate was highly active − over 100 U of FVIII per milliliter and contained virtually no VWF. This was the material we needed to immunize mice to make monoclonal antibodies to FVIII itself. When this new concentrate was run out on a gel, however, we could only see bands corresponding to the usual contaminant proteins, fibrinogen and fibronectin. But we reasoned that, with the right monoclonal to FVIII, we could devise a final purification step consisting of a column of immobilized monoclonal antiFVIII. Alison Goodall took the precious few micrograms of protein and mixed it with Freund's complete adjuvant (the infamous ‘dirty secret’ at the heart of immunology) then injected it into some mice repeatedly over 10 weeks. We tested serum from these mice for its ability to neutralize FVIII activity. One of the mice had an inhibitor titer of over 1000 BU mL−1. Following the standard Milstein method, clones were made by fusing spleen cells from this mouse with mouse myeloma cells, thus immortalizing them. To identify antiFVIII-producing cells we simply added culture supernatant from them to plasma and looked for prolongation of the clotting time. To our delight, at least 8 clonal lines of cells grew out which made potent specific antibody to human FVIII. The next step was to make enough of each antibody to test its ability to bind FVIII when it was covalently linked to sepharose. One antibody stood out – fortuitously named C8, it had high affinity and capacity. The problem was that once bound, the FVIII could not be eluted by any method we tried. However we were clearly making excellent progress with the first monoclonals to FVIII and the beginnings of a scalable purification. So the first problem was how to get the FVIII back off the C8 column. We took counsel from an expert in the new art of monoclonal antibody based immunopurification, who advised us that eluting at very high or very low pH would not work without denaturing the protein. The trick was to mix a variety of known eluting reagents, each at moderate concentration. The successful mixture that Don and Frances devised came to be known as ‘magic buffer’ and contained potassium iodide and ethylene glycol at low (but not too low) pH. As we were still working at small scale the amount of FVIII yielded was too small to visualize but the specific activity was certainly higher than ever previously reported – at least 3000 U mg−1. For the scale-up, David Heath supplied us with bulk polyelectrolyte E5 and a 20-L ‘pilot scale’ column. With Sarah Middleton's advice we set up the procedure for thawing 5 kg batches of cryoprecipitate in a stainless steel dustbin, diluting it in buffer and adding aluminum hydroxide to absorb any vitamin K-dependent proteins, centrifuging off the precipitate then pouring it down the big column packed with PE E5 over several hours. The column was washed for several more hours and eluted with a large volume of buffer, which paradoxically was concentrated by adding albumin and precipitating with polyethylene glycol. After re-dissolving this material it could then be applied to the anti VWF column. We were worried about losses from uncontrolled proteolytic activity so I revived a method I had learned in Connecticut and we added di-isopropyl-fluorophosphonate (DFP) to all the buffers and starting material. DFP is a potent inhibitor of the serine protease class of proteolytic enzymes and one of the most potent nerve gas toxins known. We were fairly cautious in its use, knowing we had enough nerve gas on hand to eliminate the population of Hampstead. We devised a protocol of wearing a full face gas mask and a disposable white suit (which was reused, as we only had one) when opening the terrifying, greasy little glass vials in a fume hood and then diluting the oily contents in buffer, with neutralizing solution nearby and antidote drawn up ready in a syringe. Apart from one near miss when a vial started to blow out and Don had to drop it instantly into the neutralizing solution, there were no actual mishaps. Between the antiVWF and antiFVIII columns, we put in an antifibronectin column to cut down that most persistent contaminant. Not having any idea about the stability of pure FVIII we, or rather Don and Frances, ran the entire procedure continuously over a 24-h period, working through the night in an atmosphere that gradually accumulated enough traces of organophosphate to give them symptoms of difficulty in breathing. Finally, we had our most concentrated sample of all, dripping out of the 10 mL antiFVIII column in ‘magic buffer’. Its specific activity recorded consistently at around 5000 U mg−1 for a final product of about 1 mg in 1 mL. But what would it look like on a polyacrylamide gel? Around that time (1980–81) a new method for ultrasensitive staining of gels with silver nitrate was described. Don set the method up and at last we could see bands that matched with bands visualized by the Western blotting method (also recently described by Towbin [3Towbin H. Staehelin T. Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proc Natl Acad Sci USA. 1979; 76: 4350-4Crossref PubMed Scopus (44924) Google Scholar]). To our great surprise, there were always multiple bands, but as each band lit up with one or other of the monoclonals to FVIII we were confident that the final product was at least 95% pure FVIII. Success at last! We also gained great confidence from the fact that the ratio of FVIII activity to antigen was unity. Many previous attempts to assess the final specific activity of FVIII had been misled by preactivation of the cofactor by thrombin; inflating the true value by up to 30-fold. The next step was to get the protein sequenced. Mike Waterfield's laboratory at the Imperial Cancer Research Fund Institute in Lincoln's Inn Fields, London, was then Britain's premier protein sequencing group and they were just starting to use a gas phase analyzer for Edman degradation. After all our hard work in making this incredibly pure and active sample it was quite distressing to see it cut up with trypsin and put down an HPLC column to derive fragments for the sequencer. It was also quite disappointing when the first runs on the new gas phase analyzer yielded no readable sequence. The first public airing of our results was at a conference devoted to FVIII under the auspices of the National Heart Lung Institute, organized by Ted Zimmerman in San Diego, California, in 1982. Everyone working on FVIII was there, together with representatives of the plasma fractionation and biotechnology industries. David Heath and I went west together, arranging to visit two of the new genetic engineering companies after the conference. The first session included presentations by each of the groups who had or thought they had achieved pure FVIII. Our paper was well received and afterwards we sat chatting with a team from Genentech including Gordon Vehar and Richard Lawn. Gordon had achieved the most convincing purification of FVIII reported up to that time, from bovine blood, in Earl Davie's laboratory and had been hired by Genentech to repeat the feat. However, the scale up had not gone well and Genentech were looking for a source of human FVIII to initiate cloning. Dick Lawn had just completed the cloning of the human albumin gene. His PhD project had been making the first large representative library of the human genome in Tom Maniatis' laboratory. Another purification reported at the conference was Ted Zimmerman and Carol Fulcher's method using sulfated dextran resin and an immobilized monoclonal to VWF. Their specific activity was about 2000 U mg−1. I was to hear more of this method later in a legal context. Dave Fass from the Mayo Clinic presented his purification of pig FVIII using a monoclonal antibody. After the congress David Heath and I visited Genentech in South San Francisco. It was my first contact with a real genetic engineering company and it convinced me that the project to clone and synthesize FVIII was not just a distant dream but actually an achievable goal in the short term. There were whole departments sequencing proteins or DNA, groups cloning human genes into expression vectors, pilot scale production and full-scale production in giant fermenters. Every laboratory was equipped with the latest scientific instruments. All this was housed in a former steel fabrication factory, a long two-story metal building with no external windows. There was a constant buzz of excitement as a new industry was being created out of genetic science. Bob Swanson, the CEO, had made the front cover of Time magazine and received a constant flow of deputations from hopeful entrepreneurs and industrialists who wanted to see how it was done. Returning via Boston, I visited the east coast biotechnology company, Biogen, that had been set up by Walter Gilbert, inventor of one of the DNA sequencing methods. A similar scene greeted me. Back in London we had to decide whom to give our FVIII to for protein sequencing. David Heath arranged for each of three biotechnology companies to make a pitch at the Sheraton Park Tower. First up was the only UK contender Celltech who presented their cloning and expression in bacteria of rennet, the calf stomach enzyme that coagulates milk and is essential for cheese making. This seemed rather far from what we were hoping to do. Biogen sent a deputation who gave impressive data on their current projects. (They later teamed with Karen Sewerin and Lars Andersson at Pharmacia.) We met the representatives of Genentech over supper who quietly outlined their plans for FVIII, which they had clearly selected as one of their company main targets and to which they had given much thought. After my visit to California, I was convinced they could do it with our input and David Heath set up the collaboration agreement. The terms were perhaps not the best that could have been achieved given the ace that we were holding but by this stage we were not the only group with pure FVIII. Royalties from synthetic FVIII sold in the Americas and Japan would accrue to Genentech and the royalties from rest of the world to Speywood and the Royal Free Medical School. With the passage of time and multiple changes of ownership this got eroded further but several million pounds eventually found their way to the Royal Free. Genentech proposed to complete the cloning, sequencing and expression in 18 months, which seemed to me over ambitious, even for them. Back in the laboratory, we set to work to make enough FVIII for Genentech's protein sequencing group. Surprisingly the protein sequencer at Genentech was using an older Beckman spinning cup machine for Edman degradation, which meant that milligram quantities of protein were required for each run. Meanwhile, Dave Fass was collaborating with Genetics Institute who had one of the prototype gas phase sequencers and only needed a few micrograms of his pig FVIII protein to get started. Altogether Don and Frances made and shipped over to Genentech 20 milligrams of FVIII, derived from 2000 L of plasma or about 10 000 blood donations. The first run yielded unique sequence from the extreme N-terminal of the protein and we knew we were on the way. For cloning purposes what would be needed was a sequence from somewhere in the protein that contained a series of amino acids with minimal code degeneracy. From that an oligonucleotide would be synthesized to label and use for screening library clones. After four further batches of FVIII had been fragmented and peptides isolated for sequencing a suitable sequence turned up − AWAYFSDVDLEK. Dick Lawn translated this into a 36mer which bound to two clones in a library specially constructed from a 4X cell line. Sequencing confirmed that the clones were authentic in that they contained DNA corresponding in sequence to the first 10 amino acids, after which an intron (number 16 as it turned out) disrupted the protein translation. From this breakthrough point Jane Gitschier set about genome walking to establish the complete gene structure and Bill Wood could start looking for a source of mRNA to do the cDNA cloning. Both turned out to be the largest cloning projects of their kind undertaken up to that time. Meanwhile we continued to make pure FVIII for further sequencing and the structure function analysis that Gordon Vehar, Bruce Kyte and Dan Eaton were doing and also for our own studies of the epitopes of the monoclonal antibodies. By the end of the project the entire protein had been sequenced in overlapping peptides, confirming the cDNA sequence, although this was never published. The news of progress was given me by weekly telephone calls to my home at night, both because of the time difference and because of the almost paranoid secrecy that surrounded the project. The idea was not to let competitors know how well it was going, so they would not be under any pressure. It was a very tense time as there are no prizes for coming second in a patent race or in academic publication. Winner takes all. At one point Bruce Kyte came over to help us with the purification and for reasons that are now lost to me rode from London to Wrexham on a motorbike with 5 mg of DFP and a kilogram of cryoprecipitate in the pannier. We were fairly relieved that he got there without accident. While Jane was walking her way through the gene in cosmid clones and Gordon was sequencing every last peptide, Bill Wood had found a lymphoid tumor cell line in a culture collection that through some accident of gene rearrangement was making FVIII mRNA. No other cell line has ever been discovered that does this and the original cell line is now lost. Gradually he pieced together the full-length cDNA clone and inserted it into an expression vector. All three of these technologically complex and demanding lines of investigation came to completion just about on target at 18 months from the arrival of the first pure protein in San Francisco, California. The day of completion was the morning that the supernatant of BHK cells transfected with the expression vector gave a positive signal in a FVIII assay, in April 1984. Genentech put in their patent applications and a few days later called a press conference, the same day that we held on
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