Albertini A, Lenfant CL, Mannucci PM, Sixma JJ (eds): Biotechnology of Plasma Proteins. Curr Stud Hematol Blood Transf. Basel, Karger, 1991, No 58, pp 46-51
Use of Recombinant Factor VIII in the Management of Hemophilia P. M. Mannucci, A. Gringeri, M. Cattaneo A. Bianchi Bonomi Hemophilia and Thrombosis Center and Institute of Internal Medicine, University of Milan, Italy
The emergence of the acquired immunodeficiency syndrome (AIDS) in hemophiliacs has added an additional and more dramatic dimension to the risks for those patients of developing blood-borne infections when they are treated with plasma-derived factor VIII (F VIII). Since 1982, when the first few cases of AIDS were reported in hemophiliacs, there have been at least four fundamental scientific advances that have decreased, if not abolished, the dramatic impact of the AIDS epidemic in hemophiliacs. These are the cloning of the F VIII gene; the availability of improved methods for the prevention of hemophilia through carrier detection and prenatal diagnosis; the development of virucidal methods which have greatly reduced the risk of infections transmitted by plasma clotting factor concentrates, and the production through recombinant DNA technology of sufficient amounts of F VIII to allow the initiation of clinical trials of this product in humans. Factor VIII
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
The relationship between structure and function of the F VIII molecule has been elucidated following F VIII cloning [ 1, 2], and is briefly summarized. In normal individuals F VIII participates, with activated F IX, calcium ions and negatively charged phospholipids, in the conversion of F X to its active form, which in turn converts prothrombin to thrombin [3-5]. Attempts to purify and characterize F VIII were rendered difficult until recently by its low concentration in plasma ( approximately 100 ng/ ml), its instability and sensitivity to proteolysis. Isolation of the gene and cDNA for F VIII was accomplished in 1984 [ 1, 2]. This was a considerable achievement since the gene was by far the largest which had been isolated and characterized. It has been estimated that transcription of the gene
Recombinant F VIII in the Management of Hemophilia
47
could take over 3 h, assuming a transcription rate at I5 nucleotides per second. The gene is 200 k long (0.1% of the X chromosome) and is contained in 26 exons, with a mRNA sequence of about 9 kb. The open reading frame encodes a primary translation product of 2,351 amino acids. The protein is organized in three discrete domains, which occur in the order Α, -Α2-B-Α3-C,-C2 [3-5]. In plasma, F VIII circulates predominantly in a metal-ion stabilized complex consisting of a variable heavy chain of 90-200 kD (representing the Α,, Α2 and B domains) and a light chain of 80 kD (representing the Α3 and C domains) [3-5]. This complex is stabilized and transported in plasma by von Willebrand factor (vWF), to which F VIII is bound in a proportion of one F VIII molecule per vWF monomer. In plasma, F VIII is proteolytically cleaved by thrombin and activated factor X. The catalytic efficiency of F VIII is greatly potentiated during these proteolytic cleavages, although unlike other coagulation factors F VIII is not converted into an enzymatically active protein [3-5]. Further exposure of F VIII to thrombin and activated protein C results in additional protein cleavages that finally inactivate the procoagulant activity of F VIII.
Two manufacturers have produced sufficient amounts of recombinant F VIII (rF VIII) to allow extensive in vitro characterization of the properties of the protein molecule and the initiation of clinical trials. The production of rF VIII by the Genetics Institute (Cambridge, Mass.) in collaboration with Baxter Hyland Division (Glendale, Calif.) was initially obtained through the expression of F VIII cDNA in mammalian cells, i.e. Chinese hamster ovarian cells. Since it was subsequently observed that co-expression of vWF cDNA in these cells resulted in the increased accumulation of two-chain F VIII into cell culture medium [6], the most recently produced batches of rF VIII are from cells engineered to express both F VIII and vWF. The rF VIII protein secreted into the cell culture medium is purified by immunoamnity chromatography with an antihuman F VIII mouse monoclonal antibody and ion-exchange chromatography, so that the final product does not contain vWF but contains trace amounts of hamster and mouse proteins. The specific activity of rF VIII produced with this method exceeds 5,000IU/mg protein but is much lower in the final vial to which plasma-derived albumin must be added to stabilize F VIII and to prevent its adsorption to glass surfaces. Intact and thrombin-treated rF VIII is similar to plasma-derived F VIII in terms of peptide and carbohydrate composition [6].
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
Biological Characteristics of Recombinant Factor VIII
Mannucci/Gringeri/Cattaneo
48
The second preparation of rF VIII now undergoing clinical trials is by Cutter Biological/Miles (Berkeley, Calif.) under a license agreement with Genentech (South San Francisco, Calif.). The product, expressed in baby hamster kidney cells, consists of multiple peptides including the 80 kD intact light chain and various extensions of the 90-kD heavy chain and contains trace amounts of hamster protein and mouse IgG from the manufacturing process [7]. The electrophoretic profiles of rF VIII on sodium dodecyl sulfate polyacrylamide gels is very similar to that of highly purified preparations of plasma-derived F VIII, with minor quantitative differences [8-10]. The carbohydrate side chains of rF VIII, contained mostly in the B domain, are similar to those of plasma F VIII [8, 9]. In vitro analysis of rF VIII demonstrates that it behaves like human F VIII in a functional clotting assay and that it can be activated and inactivated by thrombin and activated protein C like plasma-derived F VIII [7-10]. Before starting studies in humans, rF VIII was infused in congenitally F VIII-deficient dogs which closely resemble patients with hemophilia. rF VIII had full hemostatic properties in the animals, bound to circulating vWF and had normal recovery and survival in the circulation [8].
White et al. [11] were the first to report the successful use of rF VIII (Baxter Hyland) in 2 patients with severe hemophilia A. In their patients, the in vivo recovery and plasma half-life of rF VIII were similar to those obtained with plasma F VIII. After the initial infusions carried out in hospital, each patient received rF VIII at home for up to 12 months, mostly for the treatment of joint bleeding. During that time, 44 and 35 infusions, for a total of 59,572 and 37,234 F VIII units, were given to the 2 patients, respectively. The clinical response was excellent, as could be expected from the doses given, the F VIII levels attained in plasma, and the severity of bleeding episodes. There was no sign of development of alloantibodies to F VIII, nor of significant changes of tests measuring functions of the liver and kidney. Cutaneous tests of cell-mediated immunity did not improve after a year of treatment and the decline in CD4 + lymphocyte count was not affected in these 2 patients (who were both anti-HIV seropositive) [ 11]. The evidence of safety and effectiveness of rF VIII which stemmed from this phase I trial led to the organization and initiation of a larger phase II trial with the product of the same manufacturer [ 12]. The main objectives of this ongoing trial are: (1) to compare in vivo recovery and half-life of rF VIII with that of a highly purified plasma derived F VIII concentrate; (2) to confirm in a larger number of patients safety and
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
Clinical Experience with Recombinant Factor VIII
Recombinant F VIII in the Management of Hemophilia
49
tolerance of rF VIII, and (3) to establish efficacy of rF VIII in the treatment of spontaneous hemorrhages and the prevention of bleeding during and after surgery. Fifty-seven patients were enrolled so far in hemophilia centers from Europe and USA. The preliminary results of this study [ 12] demonstrate that recovery and half-life of rF VIII are not different from those of a highly purified F VIII concentrate produced from plasma by immunoamnity chromatography on monoclonal antibodies. Half-life and recovery remained stable over follow-up (up to 12 months), and only 1 patient, who had a clinical history of inhibitor development, exhibited a low-potency inhibitor (1 Bethesda units) that caused a transient decrease of F VIII recovery in vivo during treatment, but no effect on F VIII plasma half-life. Data on the use of this product in the treatment of spontaneous bleeding episodes demonstrate clearly that rF VIII is efficacious. Efficacy is also clearly shown by the performance with no mishap of 3 major surgical operations and of 6 minor operations [unpubl. observation]. There was no evidence of antibody formation towards foreign proteins of mouse and Chinese hamster possibly contaminating the preparation in trace amounts [unpubl. observation]. On the whole, these data show clearly the efficacy and safety of rF VIII for the management of acute bleeding episodes and for surgical and postoperative hemostasis. The clinical study of rF VIII produced by Cutter/Miles is organized in three phases. In phase I, the safety and pharmacokinetics of the preparation are evaluated; in phase II, the treatment is delivered at home and efficacy evaluated; surgical patients and patients requiring hospitalization for the treatment of severe hemorrhages are enrolled in phase III. Like the other study, this ongoing study is run on a multicenter, international basis, enrolling so far a total of 72 hemophiliacs from European, American and Asian centers. The report of the preliminary results [ 13] indicates that recovery and half-life of rF VIII were similar to those of an intermediate-purity plasma-derived F VIII preparation. 80% of 249 separate bleeding episodes self-managed by 40 patients at home required only one dose of rF VIII. Satisfactory hemostasis was achieved in several subjects treated in preparation for surgery or for the occurrence of severe hemorrhages requiring hospitalization. Two of 72 treated patients developed anti-F VIII antibodies, including an adult hemophiliac with a strong familial tendency for inhibitor formation and 1 of 7 previously untreated children. There has been no evidence of antibody formation to foreign proteins [unpubl.].
The preliminary clinical studies carried out with therapeutic preparations of rF VIII demonstrate that they are at least as efficacious as
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
Comments
Mannucci/Gringeri/Cattaneo
50
plasma-derived F VIII in the treatment of patients with hemophilia Α. Several questions, however, remain partially answered. It is clear that rF VIII does not carry a substantially higher risk than plasma-derived F VIII of triggering the development of F VIII alloantibodies in hemophiliacs who have been previously treated with plasma-derived F VIII. It remains to be seen, however, whether the same holds true for previously untreated patients, who carry a higher risk of developing alloantibodies. Only 7 previously untreated patients have been included so far in the Cutter study, but none as yet in the Baxter-Hyland study. Both the preparations contain human plasma albumin, which is added to the final product to stabilize F VIII and to prevent adsorption to glass vials. Although albumin seems to be a safe human protein, with little allotypic heterogeneity and contamination with other plasma proteins, it is baffling that a product such as rF VIII, made to overcome the drawbacks of plasma-derived F VIII, contains large amounts of a plasma protein. Another incompletely answered question is whether long-term treatment with rF VIII will trigger the development of antibodies to the proteins of hamster and mouse which contaminate both the preparations in trace amounts. The data obtained so far are encouraging, but longer periods of surveillance are needed. A basic question that the hemophilia community is asking is when the products will be available in amounts sufficiently large to make unnecessary the selective choice of the patients to be treated. Otherwise, on which basis shall we choose the few patients to whom rF VIII is given from the majority that will continue treatment with plasma-derived F VIII? Another question is the price. Will it be affordable or prohibitively expensive? In theory, rF VIII can be produced in unlimited amounts, with the potential to meet the needs of all the hemophiliacs, including those from the many developing countries that have at the moment no fractionation facilities and hence no plasma-derived F VIII. Finally, the impact of rF VIII on the availability of other plasma fractions for therapeutic use (albumin, immunoglobulins) which cannot be produced at the moment (and for the forseeable future), through genetic engineering remains to be clarified. Notwithstanding all these problems and issues, the availability of rF VIII, the largest plasma proteins ever cloned and produced by recombinant DNA technology, stands as a monument to human ingenuity and progress of science.
1 Toole JJ, Knopf JL, Wozney JM, et al: Molecular cloning of a cDNA encoding human antihemophilic factor. Nature 1984;312:342-347.
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
References
Recombinant F VIII in the Management of Hemophilia
51
P.M. Mannucci, MD, Via Pace 9, Ι-20122 Milano (Italy)
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
2 Wood WI, Capon DJ, Simonsen CC, et al: Expression of active human factor VIII from recombinant DNA clones. Nature 1984;312:330-336. 3 Kane WH, Davie EW: Blood coagulation factors V and VIII: Structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 1988;71:539-555. 4 Pittman DD, Kaufman Ri: Structure-function relationships of factor VIII elucidated through recombinant DNA technology. Thromb Haemost 1989;61:161-165. 5 White GC, Showmaker CB: The factor VIII gene and hemophilia A. Blood 1989;73:112. 6 Kaufman RJ, Wasley LC, Dourer AS: Synthesis, processing and secretion of recombinant human factor VIII expressed in mammalian cells. J Bío1 Chem 1988;263:63526362. 7 Klein U: Production and characterization of recombinant factor VIII. Serif Hematol 1990, in press. 8 Giles AR, Tinlin S, Hoogendoorn H, Foumel MA, Ng P, Panham N: In vivo characterization of recombinant factor VIII in a canine model of hemophilia A (factor VIII deficiency). Blood 1988;72:335-339. 9 Eaton DL, Hass PE, Riddle L, et al: Characterization of recombinant human factor VIII. J Bio! Chem 1987;262:3285-3290. 10 Fournel M: Preclinical and in vitro studies of rF VIII. Serif Herab! 1990, in press. 11 White G, McMillan CW, Kíngdon HS, Shoemaker CB: Use of recombinant antihemophilic factor in the treatment of two patients with classic hemophilia. N Engl J Med 1989;320:166-170. 12 White G, McMillan CW, Gomperts E, et al: Safety and efficacy of human factor VIII prepared by recombinant DNA techniques (abstract). Blood 1989;74(suppl 1) :55a. 13 Schwartz RS, Abildgaard C, Aledort LM, et al: Safety and efficacy of recombinantderived F VIII (rF VIII). Blood 1989;74(suppl 1):49a.
Use of Recombinant Factor VIII in the Management of Hemophilia P. M. Mannucci, A. Gringeri, M. Cattaneo A. Bianchi Bonomi Hemophilia and Thrombosis Center and Institute of Internal Medicine, University of Milan, Italy
The emergence of the acquired immunodeficiency syndrome (AIDS) in hemophiliacs has added an additional and more dramatic dimension to the risks for those patients of developing blood-borne infections when they are treated with plasma-derived factor VIII (F VIII). Since 1982, when the first few cases of AIDS were reported in hemophiliacs, there have been at least four fundamental scientific advances that have decreased, if not abolished, the dramatic impact of the AIDS epidemic in hemophiliacs. These are the cloning of the F VIII gene; the availability of improved methods for the prevention of hemophilia through carrier detection and prenatal diagnosis; the development of virucidal methods which have greatly reduced the risk of infections transmitted by plasma clotting factor concentrates, and the production through recombinant DNA technology of sufficient amounts of F VIII to allow the initiation of clinical trials of this product in humans. Factor VIII
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
The relationship between structure and function of the F VIII molecule has been elucidated following F VIII cloning [ 1, 2], and is briefly summarized. In normal individuals F VIII participates, with activated F IX, calcium ions and negatively charged phospholipids, in the conversion of F X to its active form, which in turn converts prothrombin to thrombin [3-5]. Attempts to purify and characterize F VIII were rendered difficult until recently by its low concentration in plasma ( approximately 100 ng/ ml), its instability and sensitivity to proteolysis. Isolation of the gene and cDNA for F VIII was accomplished in 1984 [ 1, 2]. This was a considerable achievement since the gene was by far the largest which had been isolated and characterized. It has been estimated that transcription of the gene
Recombinant F VIII in the Management of Hemophilia
47
could take over 3 h, assuming a transcription rate at I5 nucleotides per second. The gene is 200 k long (0.1% of the X chromosome) and is contained in 26 exons, with a mRNA sequence of about 9 kb. The open reading frame encodes a primary translation product of 2,351 amino acids. The protein is organized in three discrete domains, which occur in the order Α, -Α2-B-Α3-C,-C2 [3-5]. In plasma, F VIII circulates predominantly in a metal-ion stabilized complex consisting of a variable heavy chain of 90-200 kD (representing the Α,, Α2 and B domains) and a light chain of 80 kD (representing the Α3 and C domains) [3-5]. This complex is stabilized and transported in plasma by von Willebrand factor (vWF), to which F VIII is bound in a proportion of one F VIII molecule per vWF monomer. In plasma, F VIII is proteolytically cleaved by thrombin and activated factor X. The catalytic efficiency of F VIII is greatly potentiated during these proteolytic cleavages, although unlike other coagulation factors F VIII is not converted into an enzymatically active protein [3-5]. Further exposure of F VIII to thrombin and activated protein C results in additional protein cleavages that finally inactivate the procoagulant activity of F VIII.
Two manufacturers have produced sufficient amounts of recombinant F VIII (rF VIII) to allow extensive in vitro characterization of the properties of the protein molecule and the initiation of clinical trials. The production of rF VIII by the Genetics Institute (Cambridge, Mass.) in collaboration with Baxter Hyland Division (Glendale, Calif.) was initially obtained through the expression of F VIII cDNA in mammalian cells, i.e. Chinese hamster ovarian cells. Since it was subsequently observed that co-expression of vWF cDNA in these cells resulted in the increased accumulation of two-chain F VIII into cell culture medium [6], the most recently produced batches of rF VIII are from cells engineered to express both F VIII and vWF. The rF VIII protein secreted into the cell culture medium is purified by immunoamnity chromatography with an antihuman F VIII mouse monoclonal antibody and ion-exchange chromatography, so that the final product does not contain vWF but contains trace amounts of hamster and mouse proteins. The specific activity of rF VIII produced with this method exceeds 5,000IU/mg protein but is much lower in the final vial to which plasma-derived albumin must be added to stabilize F VIII and to prevent its adsorption to glass surfaces. Intact and thrombin-treated rF VIII is similar to plasma-derived F VIII in terms of peptide and carbohydrate composition [6].
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
Biological Characteristics of Recombinant Factor VIII
Mannucci/Gringeri/Cattaneo
48
The second preparation of rF VIII now undergoing clinical trials is by Cutter Biological/Miles (Berkeley, Calif.) under a license agreement with Genentech (South San Francisco, Calif.). The product, expressed in baby hamster kidney cells, consists of multiple peptides including the 80 kD intact light chain and various extensions of the 90-kD heavy chain and contains trace amounts of hamster protein and mouse IgG from the manufacturing process [7]. The electrophoretic profiles of rF VIII on sodium dodecyl sulfate polyacrylamide gels is very similar to that of highly purified preparations of plasma-derived F VIII, with minor quantitative differences [8-10]. The carbohydrate side chains of rF VIII, contained mostly in the B domain, are similar to those of plasma F VIII [8, 9]. In vitro analysis of rF VIII demonstrates that it behaves like human F VIII in a functional clotting assay and that it can be activated and inactivated by thrombin and activated protein C like plasma-derived F VIII [7-10]. Before starting studies in humans, rF VIII was infused in congenitally F VIII-deficient dogs which closely resemble patients with hemophilia. rF VIII had full hemostatic properties in the animals, bound to circulating vWF and had normal recovery and survival in the circulation [8].
White et al. [11] were the first to report the successful use of rF VIII (Baxter Hyland) in 2 patients with severe hemophilia A. In their patients, the in vivo recovery and plasma half-life of rF VIII were similar to those obtained with plasma F VIII. After the initial infusions carried out in hospital, each patient received rF VIII at home for up to 12 months, mostly for the treatment of joint bleeding. During that time, 44 and 35 infusions, for a total of 59,572 and 37,234 F VIII units, were given to the 2 patients, respectively. The clinical response was excellent, as could be expected from the doses given, the F VIII levels attained in plasma, and the severity of bleeding episodes. There was no sign of development of alloantibodies to F VIII, nor of significant changes of tests measuring functions of the liver and kidney. Cutaneous tests of cell-mediated immunity did not improve after a year of treatment and the decline in CD4 + lymphocyte count was not affected in these 2 patients (who were both anti-HIV seropositive) [ 11]. The evidence of safety and effectiveness of rF VIII which stemmed from this phase I trial led to the organization and initiation of a larger phase II trial with the product of the same manufacturer [ 12]. The main objectives of this ongoing trial are: (1) to compare in vivo recovery and half-life of rF VIII with that of a highly purified plasma derived F VIII concentrate; (2) to confirm in a larger number of patients safety and
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
Clinical Experience with Recombinant Factor VIII
Recombinant F VIII in the Management of Hemophilia
49
tolerance of rF VIII, and (3) to establish efficacy of rF VIII in the treatment of spontaneous hemorrhages and the prevention of bleeding during and after surgery. Fifty-seven patients were enrolled so far in hemophilia centers from Europe and USA. The preliminary results of this study [ 12] demonstrate that recovery and half-life of rF VIII are not different from those of a highly purified F VIII concentrate produced from plasma by immunoamnity chromatography on monoclonal antibodies. Half-life and recovery remained stable over follow-up (up to 12 months), and only 1 patient, who had a clinical history of inhibitor development, exhibited a low-potency inhibitor (1 Bethesda units) that caused a transient decrease of F VIII recovery in vivo during treatment, but no effect on F VIII plasma half-life. Data on the use of this product in the treatment of spontaneous bleeding episodes demonstrate clearly that rF VIII is efficacious. Efficacy is also clearly shown by the performance with no mishap of 3 major surgical operations and of 6 minor operations [unpubl. observation]. There was no evidence of antibody formation towards foreign proteins of mouse and Chinese hamster possibly contaminating the preparation in trace amounts [unpubl. observation]. On the whole, these data show clearly the efficacy and safety of rF VIII for the management of acute bleeding episodes and for surgical and postoperative hemostasis. The clinical study of rF VIII produced by Cutter/Miles is organized in three phases. In phase I, the safety and pharmacokinetics of the preparation are evaluated; in phase II, the treatment is delivered at home and efficacy evaluated; surgical patients and patients requiring hospitalization for the treatment of severe hemorrhages are enrolled in phase III. Like the other study, this ongoing study is run on a multicenter, international basis, enrolling so far a total of 72 hemophiliacs from European, American and Asian centers. The report of the preliminary results [ 13] indicates that recovery and half-life of rF VIII were similar to those of an intermediate-purity plasma-derived F VIII preparation. 80% of 249 separate bleeding episodes self-managed by 40 patients at home required only one dose of rF VIII. Satisfactory hemostasis was achieved in several subjects treated in preparation for surgery or for the occurrence of severe hemorrhages requiring hospitalization. Two of 72 treated patients developed anti-F VIII antibodies, including an adult hemophiliac with a strong familial tendency for inhibitor formation and 1 of 7 previously untreated children. There has been no evidence of antibody formation to foreign proteins [unpubl.].
The preliminary clinical studies carried out with therapeutic preparations of rF VIII demonstrate that they are at least as efficacious as
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
Comments
Mannucci/Gringeri/Cattaneo
50
plasma-derived F VIII in the treatment of patients with hemophilia Α. Several questions, however, remain partially answered. It is clear that rF VIII does not carry a substantially higher risk than plasma-derived F VIII of triggering the development of F VIII alloantibodies in hemophiliacs who have been previously treated with plasma-derived F VIII. It remains to be seen, however, whether the same holds true for previously untreated patients, who carry a higher risk of developing alloantibodies. Only 7 previously untreated patients have been included so far in the Cutter study, but none as yet in the Baxter-Hyland study. Both the preparations contain human plasma albumin, which is added to the final product to stabilize F VIII and to prevent adsorption to glass vials. Although albumin seems to be a safe human protein, with little allotypic heterogeneity and contamination with other plasma proteins, it is baffling that a product such as rF VIII, made to overcome the drawbacks of plasma-derived F VIII, contains large amounts of a plasma protein. Another incompletely answered question is whether long-term treatment with rF VIII will trigger the development of antibodies to the proteins of hamster and mouse which contaminate both the preparations in trace amounts. The data obtained so far are encouraging, but longer periods of surveillance are needed. A basic question that the hemophilia community is asking is when the products will be available in amounts sufficiently large to make unnecessary the selective choice of the patients to be treated. Otherwise, on which basis shall we choose the few patients to whom rF VIII is given from the majority that will continue treatment with plasma-derived F VIII? Another question is the price. Will it be affordable or prohibitively expensive? In theory, rF VIII can be produced in unlimited amounts, with the potential to meet the needs of all the hemophiliacs, including those from the many developing countries that have at the moment no fractionation facilities and hence no plasma-derived F VIII. Finally, the impact of rF VIII on the availability of other plasma fractions for therapeutic use (albumin, immunoglobulins) which cannot be produced at the moment (and for the forseeable future), through genetic engineering remains to be clarified. Notwithstanding all these problems and issues, the availability of rF VIII, the largest plasma proteins ever cloned and produced by recombinant DNA technology, stands as a monument to human ingenuity and progress of science.
1 Toole JJ, Knopf JL, Wozney JM, et al: Molecular cloning of a cDNA encoding human antihemophilic factor. Nature 1984;312:342-347.
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
References
Recombinant F VIII in the Management of Hemophilia
51
P.M. Mannucci, MD, Via Pace 9, Ι-20122 Milano (Italy)
Downloaded by: Université de Paris 193.51.85.197 - 1/12/2020 2:12:16 AM
2 Wood WI, Capon DJ, Simonsen CC, et al: Expression of active human factor VIII from recombinant DNA clones. Nature 1984;312:330-336. 3 Kane WH, Davie EW: Blood coagulation factors V and VIII: Structural and functional similarities and their relationship to hemorrhagic and thrombotic disorders. Blood 1988;71:539-555. 4 Pittman DD, Kaufman Ri: Structure-function relationships of factor VIII elucidated through recombinant DNA technology. Thromb Haemost 1989;61:161-165. 5 White GC, Showmaker CB: The factor VIII gene and hemophilia A. Blood 1989;73:112. 6 Kaufman RJ, Wasley LC, Dourer AS: Synthesis, processing and secretion of recombinant human factor VIII expressed in mammalian cells. J Bío1 Chem 1988;263:63526362. 7 Klein U: Production and characterization of recombinant factor VIII. Serif Hematol 1990, in press. 8 Giles AR, Tinlin S, Hoogendoorn H, Foumel MA, Ng P, Panham N: In vivo characterization of recombinant factor VIII in a canine model of hemophilia A (factor VIII deficiency). Blood 1988;72:335-339. 9 Eaton DL, Hass PE, Riddle L, et al: Characterization of recombinant human factor VIII. J Bio! Chem 1987;262:3285-3290. 10 Fournel M: Preclinical and in vitro studies of rF VIII. Serif Herab! 1990, in press. 11 White G, McMillan CW, Kíngdon HS, Shoemaker CB: Use of recombinant antihemophilic factor in the treatment of two patients with classic hemophilia. N Engl J Med 1989;320:166-170. 12 White G, McMillan CW, Gomperts E, et al: Safety and efficacy of human factor VIII prepared by recombinant DNA techniques (abstract). Blood 1989;74(suppl 1) :55a. 13 Schwartz RS, Abildgaard C, Aledort LM, et al: Safety and efficacy of recombinantderived F VIII (rF VIII). Blood 1989;74(suppl 1):49a.
