Mammalian recombinant proteins: structure, function and immunological analysis Randal I. Kaufman Genetics Institute, 87 Cambridge Park Drive, Cambridge, M A 02140, USA Current Opinion in Biotechnology 1990, 1:141-150

Introduction Recombinant DNA technology has provided a means of developing new protein pharmaceuticals with wide ranging activities. In addition, it has provided an approach for studying protein structure and function with the hope of increasing or otherwise modifying the biological activities of these proteins to derive improved therapeutic agents. The diversity of these proteins and the wide range of their post-translational modifications has added complexity to the evaluation of their in vivo efficiency and potential immunogenicity. Because many proteins require specific post-translational modifications for biological activity, mammalian cells have become a choice host for their production. The therapeutics produced from mammalian cells include blood coagulation factors such as Factor VIII for treatment of hemophilia A, fibrinolytic agents such as tissue plasminogen activator (tPA), and erythropoietin (EPO) for treatment of patients with chronic anemia. In addition, various interferons and interleukins, as well as granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor (GM-CSF) are being used in clinical trials in cancer patients and bone marrow transplant recipients. This review will address the production of such proteins and the approaches taken to improve their therapeutic value. In general, the focus of the review is directed towards hemophilia A because a large amount of accumulated information is available on the therapeutic usefulness of plasma-derived Factor VIII and the immunological response. Particular emphasis will be placed on the problems of obtaining Factor VIII with the post-translational modifications that are required for, or otherwise influence, functional activity. The review will discuss other examples of potentially useful proteins produced and designed by recombinant DNA technology as they relate to the concepts presented.

Protein synthesis in mammalian cells Mammalian cells are often used as hosts for the expression of human genes because they are usually able

to recognize the signals for synthesis, processing, and secretion of the protein products. In these systems, it has been demonstrated that heterologous proteins are readily synthesized and secreted into the growth medium. In addition, the protein folding and disulfide bond formation are usually like that of the natural protein, and many post-translational modifications can occur. In many cases, these modifications are required for, or otherwise alter, the biological activity and/or immunogenicity of the derived product. The highly compartmentalized structure of eukaryotic cells requires a mechanism for proteins to be directed to the appropriate organelle such as the endoplasmic reticulum (ER). The precursor forms of proteins transported into the ER generally conrain a hydrophobic signal sequence at or near the amino terminus. These proteins are cotranslationally translocated into the lumen of the ER and the signal sequence is usually cleaved by signal peptidase during this process. As the protein enters the ER, asparagine (N)-linked glycosylation occurs at appropriate recognition sites (Asn-X-Ser/Thr). The use of particular N-linked sites is determined by the structure of the growing polypeptide backbone which means that proteins expressed in heterologous cells frequently exhibit occupancy of N-linked sites very similar to that of the native polypeptide. As the protein is translocated into the lumen of the ER, it undergoes a complex series of reactions to achieve its final conformation. These steps include: protein folding; disulphide bond formation; oligomerization of multisubunit proteins; hydroxylation of specific amino acid residues such as proline, aspartic acid, asparagine, and lysine; trimming of glucose and mannose residues from the core N-linked oligosaccharides; and fatty acid acylation. The culmination of these steps is the vesicle-mediated transport of the polypeptide from the ER to the c/s-Golgi compartment. As the protein travels through the Golgi complex, further modifications occur. These include the addition of N-acetylglucosamine, galactose, and sialic acid to N-linked carbohydrate; serine and threonine O-linked glycosylation; sulfation of tyrosine and carbohydrate residues; phosphorylation and cleavage of propolypeptides. In addition, vitamin K-dependent 7-carboxylation of glutamic acid residues occurs (although the

Abbreviations APC--activated protein C; CHO--Chinese hamster ovary; EPO~erythropoietin; ER-~endoplasmic reticulum; GM-CSF~granulocyte macrophage colony-stimulating factor; PACE~paired-basic amino acid-cleaving enzyme; TGF~transforming growth factor; tPA--tissue plasminogen activator; vWF~von Willebrand factor. (~ Current Biology Ltd ISSN 0958-1669

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Mammalian gene studies precise location of this modification has not been identified). In some cell types, secretory proteins may enter a regulated secretory pathway and become stored in vesicles for release upon stimulation. This regulated pathway appears to be required for the maturation of certain precursor polypeptides such as the processing of proinsulin to insulin and proopiomelanocortin to adrenocorticotropic hormone, [3-melanocyte stimulating hormone and endorphins. The multitude of complex post-translational modifications that many secreted proteins undergo has provided a major challenge - - the development of efficient expression systems in mammalian cells that will produce proteins with high specific biological activity and with post-translational modifications that are sufficiently like those on the natural protein to avoid any immune response to the recombinant protein when it is used therapeutically.

Development of recombinant Factor VIII for treatment of hemophilia A Hemophilia A is a bleeding disorder caused by a deftciency or abnormality in the blood clotting protein Factor VIII. Upon damage to a blood vessel wall, platelets adhere to the subendothelium, aggregate, and become activated. The surfaces of these activated platelets assemble protease complexes, which results in the sequential activation of a series of vitamin K-dependent coagulation factors; Factor VII, Factor IX, Factor X, and prothrombin. Factor VIII functions in the intrinsic pathway of coagulation as a cofactor for the activated Factor IX (IXa)-mediated activation of Factor X. In the presence of negatively charged phospholipid and calcium, Factor VIII acts to increase 10 000-fold the Vmax of the Factor IXa-mediated activation of Factor X. The mechanism by which Factor VIII functions in the Factor Xa-generating complex is poorly understood. By contrast, the mechanism by which the homologous cofactor, Factor V, functions in the thrombin-generating complex has been extensively studied. The association of Factor Va with Factor Xa on a phospholipid surface induces an allosteric conformational change in the active site of Factor Xa which positions it at a distance above the membrane for optimal prothrombinase activity [1 °]. As Factor VIII shares functional as well as structural similarity with Factor V [1 o], the mechanism by which Factor VIIIa accelerates the proteolysis of Factor X by Factor IXa may also involve the binding of Factor VIIIa and Factor IXa on a phospholipid surface to facilitate a conformational change that favors catalysis. The cloning of the human Factor VIII gene [2,3] and its expression in mammalian cells [4 °] has enhanced our understanding of the protein structure, biosynthesis, and post-translational ~odifications that are important for its function. In addition, the structural requirements for Factor VIII function have been elucidated through site-directed DNA-mediated mutagenesis [5 °].

In plasma, Factor VIII circulates as a heterodimer composed of a heavy chain extending up to 200 kD in a metal ion association with an 80 kD light chain. This heterodimer is non-covalently bound to another high molecular weight glycoprotein, von Willebrand factor (vWF), which functions in hemostasis to mediate the interaction of platelets to the damaged subendothelium. In plasma, vWF is required to stabilize Factor VIII. It may also serve to protect Factor VIII from proteolysis by activated Factor X and activated protein C (APC). The deduced primary amino acid sequence of human Factor VIII determined from the cloned cDNA revealed a domain structure of AI-.A2:B'~_3:Cl:C2 (Fig. 1). The A domains of Factor VIII occur twice in the heavy chain and once in the light chain and have 30% homology to each other and to the A domains of ceruloplasmin, a copper-binding plasma protein. This suggests that the A domains of Factor VIII may be involved in metal ion binding. These domains also exhibit 40% homology to the A domains of Factor V. The C domains occur twice in the carboxy terminus of the Factor VIII light chain and exhibit homology to phospholipid-binding proteins, suggesting that they may have a role in phospholipid interaction. The B domain is encoded by a single large exon of 3100 nucleotides, has no known homology to other proteins, and contains 18 out of the 25 potential N-linked glycosylation sites within Factor VIII. Analysis of the biosynthesis of Factor VIII expressed from the cloned gene introduced into Chinese hamster ovary (CHO) cells identified a number of post-translocational modifications of the primary translation product [4 o] (Fig. 2). Factor VIII is synthesized as a 2351 amino acid single chain precursor. Upon translocation into the lumen of the ER, a 19-amino-acid signal peptide is cleaved from this and addition of N-linked high mannose oligosacchafides occurs. In the ER the majority of Factor VIII interacts with a resident heavy-chain binding protein, BiP (or glucose-regulated protein 78). The Factor VIII bound to BiP requires high levels of ATP for its release and efficient secretion, which is in contrast with other proteins that exhibit a lesser interaction with BiP [6 ° "]. Studies on the role of BiP in secretion suggest that it serves a retention function, possibly to ensure 'quality control' for proteins that are secreted from the cell [6 ° °,7 ° °]. The secretion-competent Factor VIII travels to the Golgi apparatus where the majority of Factor VIII is cleaved at two sites after residues 1313 and 1648 to generate amino-terminal-derived heavy chains extending up to 200 kD and a carboxy-terminal-defived light chain of 80 kD. Still within the Golgi apparatus, Factor VIII is further processed by: modifications of the N-linked high mannose oligosacchafides to complex types; addition of carbohydrate to serine and threonine residues; and addition of sulfate to specific tyrosine residues within the heavy and light chains. Upon secretion from the cell, Factor VIII is detected as a metal ion-associated complex of the heavy and light chain fragments.

Mammalian recombinant proteins: structure, function and immunological analysis Kaufman

500

Amino acid

N"21

1000

I

al

H

I

1500

I

A2

I

I

A3

200kD

M

2000

I

I

H

I cl I c2 I COOH

80kD

I COOH

IIA ~ 740

[

90~

kD

;372 Xa~336

I[

~i

.c.

]

11a

I 50kD Ila, APE:,

43kD

I

I 45kD ID

H

80kD

I

D[

73kD

]

11a~1689 Xa~1721 [

67kD

I

Fig. 1. Domain structure and proteolytic processing of Factor VIII. The structural

domains of Factor VIII deduced from the primary amino acid sequence are shown. The A domains span approximately 300 amino acids and are repeated three times. The two C domains span 150 amino acids and the B domain spans 980 amino acids. Intracellularly, Factor VIII is cleaved within the B domain to generate a 200 kD heavy chain polypeptide and an 80 kD light chain polypeptide. The thrombin (lla) cleavage products are shown. APC, activated protein C; HCR, heavily glycosylated region.

Proteolytic processing requirements for Factor VIII function Mutagenesis of arginine residues to isoleucines on the amino-terminal side of the cleavage sites at 1313 and 1648 results in the secretion of Factor VIII as a single-chain polypeptide. This was demonstrated to be fully functional [8]. The cleavages that occur within Factor VIII are typical of many propeptides. Cleavage sites are usually marked by paired basic amino acid residues (Arg-Arg or Lys-Arg) or by a monobasic residue with an. arginine at the - 4 position. In contrast to Factor VIII, many propeptides require appropriate cleavage to mediate the processes necessary for functional activity. Examples of these processes are: protein folding and disulfide bond formation in insulin and vWF [9]; 7-carboxylation of glutamic acid residues in vitamin K coagulation factors, such as Factor IX [10]; intracellular targeting of proteins such as prosomatostatin [11 -]; and the coordinate synthesis of multiple mature peptides from a single precursor polypeptide (e.g. the cleavage of proopiomelanocortin to yield seven neuroendocrine peptide hormones [12]). In addition, many viral polypeptides require cleavage of sites after paired basic amino acid residues for viral infectivity. Examples are the human immunodeficiency virus envelope gene product gpl60 [13 .,14], and several polypro-

teins encoded by members of the herpes vires family [15]. Propolypeptides expressed in mammalian cells are usually cleaved correctly. At high expression levels, however, the propeptide cleaving activity may become saturated [9,16 -,17 .]. The enzyme(s) responsible for the processing of these precursors have not been fully characterized at the molecular level. The recent identification [18 • "] of a human homologue of the subtilisin-like serine protease encoded by the yeast Kex2 gene is the first reported isolation of a gene encoding a functional human propeptide processing enzyme. Oveiexpression of the cDNA for this enzyme which has been called pairedbasic amino acid-cleaving enzyme (PACE), can improve the processing of pro-vWF expressed in mammalian cells [18 • .]. Overexpression of PACE provides a means to improve propeptide processing of recombinant proteins expressed in mammalian cells. An altemative method to improve propeptide processing involves mutagenesis of cleavage sites to make them more ettlcient substrates for the cleaving activity [16 .,17 -]. In addition to intracellular proteotytic processing, Factor VIII is proteolytically activated by thrombin and Factor Xa. Proteolysis of both the heavy and light chains of Factor VIII coincides with a rapid increase in procoagulant

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Mammalian

gene studies Fig. 2. Synthesis, processing and secretion of Factor VIII in mammalian cells. The Factor VIII primary translation product is translocated into the lumen of the endoplasmic reticulum (ER) where N-linked glycosylation occurs ( I ) . A fraction of Factor VIII binds tightly to BiP and is probably destined for degradation. A proportion of the molecules travel to the Golgi where the following complex modifications occur: addition of N-linked high mannose oligosaccharides ( } ) ; modification of carbohydrate on N-linked sites (~); addition of carbohydrate to serine and threonine residues ( I ); sulfation of t yrosine residues; and cleavage of the protein to the mature heavy and light chains. The presence of von Willebrand factor (vWF) in the medium promotes secretion of stable Factor VIII. In the absence of vWF, the individual chains are secreted and degraded. The A ([]), B ([]), and C (~) domains are indicated. The acidic regions where tyrosine sulfation occurs adjacent to the A domains are shown (solid black). Me denotes metal ion. Adapted from [4 °] with permission.

-vWF

+vWF

Extracellular medium I

I I

activity (Fig. 1). Cleavage within the heavy chain after arginine residue 740 generates a 90 kD polypeptide which is subsequently cleaved after arginine residue 372 to generate polypeptides of 50 kD and 43 kD. Concomitantly, the 80 kD light dhain is cleaved after arginine residue 1689 to generate a 73 kD polypeptide. Site-directed DNAmediated mutagenesis has been used to mutate individually every cleavage site within Factor VIII [8]. The only mutations that inhibited thrombin activation were arginine to isoleucine changes at either residue 372 or 1689.

In both cases, the resulting molecules were not cleaved by thrombin at the mutated site and were not, susceptible to thrombin activation. It has been suggested that cleavage at residue 1689 is required to release Factor VIII from vWF [19]. The crucial importance of cleavage at residues 372 and 1689 for activation of Factor VIII is also evident from the identification of hemophilia A patients who have missense mutations at either of these sites [20,21]. These patients have normal levels of circulating Factor VIII antigen but no detectable Factor VIII activity.

Mammalian recombinant proteins: structure, function and immunological analysis Kaufman After activation, there is a subsequent first order decay of Factor VIII activity. The mechanism by which this loss in procoagulant activity occurs is poorly understood. It is possible that once Factor VIII is activated it. undergoes a conformational change that allows the chains to dissociate [22]. Factor VIII and Factor ViiIa can be inactivated by APC-mediated cleavage within the heavy chain at arginine residue 336 [23], whereas a binding site for APC has been localized within the 80 kD light chain within domain A3 [24 ° -]. Factor Xa and thrombin can also be cleaved after residue 336 [23]. Site-directed mutagenesis of arginine 336 to an isoleucine yields a Factor VIII molecule of increased specific activity, possibly because the molecule has gained resistance to proteolytic inactivation in the clotting assay [9]. An understanding of the mechanism of inactivation may provide the means of deriving longer-acting derivatives of Factor VIII for therapeutic use. In addition to Factor VIII, numerous other clotting factors require cleavage for activation. It has been possible to engineer forms of activated factors by introducing intracellular processing sites within the polypeptide. These mutant proteins become cleaved and activated upon secretion from the cell. This approach has been successfully applied to the production of human APC by the introduction of an insulin receptor precursor cleavage site at the protein C site for thrombin-mediated activation [25 • "].

Role of carbohydrate for functional activity The carbohydrate chains of a glycoprotein may affect its confirmation leading to changes in its intracellular transport and secretion, solubility, susceptibility to proteases, in vivo half life, biological activity, or immunogenicity. The role of glycosytation has been examined by treatment of proteins with glycosidases, synthesis of proteins in the presence of inhibitors of carbohydrate addition, expression of proteins in different cell types or cell mutants with defects in any of the glycosyltransferases, and with mutant proteins produced by directed mutagenesis of sites for carbohydrate addition [26,27 ",28,29, 30 • ",31 • "]. Analysis of the carbohydrate added to Factor VIII expressed in CHO cells has demonstrated that both the occupancy and complexity of the N-linked sites are very much like that of the natural plasma-derived protein [4 .]. As 18 out of the 25 N-linked sites are located within t h e B domain of Factor VIII, site-directed DNA-mediated mutagenesis to delete the B domain prorides a means of determining whether this heavily glycosylated domain is essential to the function of Factor VIII. Molecules with deleted B domains are observed to be more efficiently secreted from the cell [32]. Detailed analysis reveals that Factor VIII molecules without domain B exhibit a reduced interaction with BiP, but are similar to the wild-type molecule with respect to specific activities, thrombin cleavage products, and thrombin activation coefficients [33-35]. After infusion into a Factor VIII-deficient dog, the B-domain deletion molecules exhibit no

detectable differences from wild-type Factor VIII in vWF binding, survival in plasma, or ability to normalize the cuticle bleeding time [34,35]. These analyses demonstrate that removal of the heavily glycosylated B domain does not affect in vitro or in vivo procoagulant activity. However, the functional significance of the B domain remains unknown. Although both the amino acid sequences of the A and C domains of Factor V are 40% homologous to the A and C domains of Factor VIII, the two B domains are unrelated in sequence. The most significant common feature of the two B domains is their large content of both N-linked- and serine/threonine O-linked-oligosaccharides. Both B domains are cleaved from the molecule upon cofactor activation. As Factors VIII and V are likely to have evolved through gene duplication and divergence, it is possible that the conserved heavy glycosylation is of functional significance. One difference between the wildtype and B-domain-deleted Factor VIII is that when purified away from vWF, the latter exhibits greater sensitivity to thrombin activation [34]. This suggests that the B domain may protect Factor VIII from thrombin activation, perhaps by steric hindrance caused by the carbohydrate groups present within the domain. In this respect, it is of interest that the activation peptides of many coagulation factors are heavily glycosylated. Although it appears that the heavily glycosylated B domain is not essential for Factor VIII activity, examples from other recombinant proteins suggest that carbohydrate may dramatically influence important properties of molecules. Thus, occupancy by oligosaccharides of the N-linked glycosylation site (Asn184) between the two kringle structures of tP& slows the rate of plasmin-catalyzed conversion to two-chain tPA [36 "]. Mutagenesis of the N-linked glycosylation sites of tPA yields results which suggest that glycosylation may influence fibrin binding, fibrinolytic, and fibrinogenolytic a ctivities [26]. Also, the N-linked Oligosaccharides of trailsforming growth factor (TGF)-131 have been shown to play a role in the maintenance of precursor latency [27 "]. In many cases deglycosylation of growth factors results in higher atFmity for their receptor; this has been shown for GM-CSF [28] and EPO [29]. Most significantly, EPO conrains a rare N-linked oligosaccharide which contains polylactosamine [Gal([31,4)-GlcNAc(131,3)-] repeats. Both recombinant EPO and naturally derived EPO exhibit this unusual moiety on the tetra-antennary branched N-linked oligosaccharides. Takeuchi et al. [30 • "] reported that CHO-derived EPO with biantennary structure was sevenfold less active in vivo compared with the tetra-antennary forms that contain polylactosamine. However, there was no difference between the in vitro biological activities of the two types of EPO molecules. Similar results were obtained in studies using EPO produced in cells that were defective in the addition of galactose to the growing oligosaccharide [31 • "]. EPO with incompletely processed N-linked oligosaccharides was fully functional in vitro but lacked in vivo biological activity. Only a portion of the loss of activity could be accounted for by a rapid clearance of the EPO. These results taken together suggest that the carbohydrate moieties of EPO may not be

145

146 Mammalian gene studies involved in receptor binding but are important in other interactions required for in vivo activity.

Post-translational addition of sulfate to tyrosine is required for Factor VIII procoagulant activity A large number of plasma proteins contain sulfated tyrosine residues [37]. Interestingly, many of these proteins, such as hirudin, fibrinogen, heparin cofactor II, a2-antiplasmin, vitronectin, and Factor V, interact with thrombin. Sulfated tyrosine in hirudin occurs in a region that forms electrostatic interactions with thrombin [38 ° °]. The process of tyrosine sulfation increases the affinity of hirudin for thrombin at least 10-fold [39]. A comparison of proteins containing tyrosine sulfate has led to the identification of consensus amino acids that act as 'signatures' for tyrosine sulfation [37]. This process is mediated by the tyrosylprotein sulfotransferase localized in the trans-Golgi apparatus [40]. Several tyrosines within Factor VIII conform to these consensus features. Direct analysis by amino acid sequencing of peptide fragments has demonstrated the existence of sulfated tyrosine residues within the heavy chain at residue 346 and within the light chain at residues 1664 and 1680 [41]. In addition, the C-terminal half of the 90 kD thrombin fragment of the heavy chain contains one or more tyrosine residues at positions 718, 719, and 723. It is of interest that these sulfated tyrosine residues are present at the border of the thrombin cleavage sites, suggesting that they may have functional importance in thrombin interaction. The functional importance of the tyrosine sulfate within Factor VIII has been examined both by studying Factor VIII synthesized in the presence of sodium chlorate, a potent inhibitor of tyrosine sulfation, and by site-directed DNA-mediated mutagenesis of the tyrosine residues to phenylalanine residues (a conservative change) in order to inhibit sulfation [41]. As sulfate incorporation was increasingly inhibited by the addition of sodium chlorate to Factor VIII-producing cells, there was a proportional reduction in the activity of the secreted Factor VIII. However, the presence of sodium chlorate did not affect the synthesis or secretion of Factor VIII antigen. These resuits suggest tyrosine sulfation is not required for Factor VIII synthesis and secretion, but is an important requirement for its procoagulant activity. Results from the mutagenesis studies demonstrated that a tyrosine-to-phenylalanine change at any single site did not alter the specific activity of the expressed Factor VIII. The procoagulant activity and thrombin cleavage of the secreted Factor VIII molecules harboring any of the single tyrosineto-phenylalanine mutations are not significantly altered. This suggests that no single tyrosine sulfation site is essential for t h r o m b ~ activation and/or activity. The finding that sodium chlorate inhibited Factor VIII activity but that individual mutations of tyrosine residues which are sulfated did not may indicate that functional Factor VIII activity requires sulfation on at least two tyrosine residues. The tyrosine-to-phenylalanine mutation at

residue 1680 of the light chain did reduce affinity for vWF. These results are consistent with the observations that a monoclonal antibody that recognizes aspartic acid 1679 and tyrosine 1680 can inhibit interaction of Factor VIII with CWF [42] and that deletion of this region within the Factor VIII molecule destroys vWF binding properties [8]. These experiments suggest that post-translational sulfation of tyrosine residues in the light chain of Factor VIII may contribute to stabilizing the Factor VIII-vWF interaction. To date there are very few examples where sulfation of either tyrosine or carbohydrate is known to influence biological activity. Exceptions are the gonadotropic hormones - - luteinizing hormone, folliclestimulating hormone, and thyroid-stimulating hormone - - which exhibit different activities depending on their degree of carbohydrate sulfation [43]. Additionally, sulfation of tyrosine is required within cholecystokinin for its activity [44] and within hirudin for maximum affinity towards thrombin [39 °].

Immunogenicity of recombinant proteins The immune system has the potential to differentiate between very small differences in polypeptide composition within a protein. For example, it has long been established that porcine insulin is capable of eliciting anti-insulin antibodies in humans. Additionally, the production of antibodies that react with unused O-linked glycosylation sites within human GM-CSF has now been reported [45 ° °]. Immunogenicity of recombinant proteins is of particular concern when evaluating the efficiency of protein therapeutic agents, particularly those that are administered chronically. Some antibodies that are produced may react only with the recombinant protein. In other cases, antibodies may not only react with the recombinant protein but also crossreact with the natural protein. Of greatest concern are situations where antibodies are produced that neutralize the activity of the recombinant and/or natural protein. Non-neutralizing antibodies may alter the plasma clearance of the protein; for example, antibodies to cz-interferon were found to reduce its renal clearance [46]. For the majority of recombinant DNA products in therapeutic use there has not been a detailed analysis of the antigenic epitopes responsible for the immune response. The definition of antigenic epitopes is likely to be a critical area for future clinical studies. Knowledge has accumulated concerning the anti-Factor VIII and anti-Factor IX antibodies generated in hemophiliacs treated with plasma-derived preparations of Factor VIII or Factor IX. In hemophilia B patients, the development of inhibitory antibodies is closely correlated with deletion of the Factor IX gene, whereas in hemophilia A patients the correlation with Factor VIII gene deletion is not so good. The reason may be that in the majority of cases where the Factor VIII gene is defective rather than deleted, no crossreactive Factor VIII is present in the plasma, whereas many Factor IX gene defects result in the presence of immunologically crossreactive Factor IX in the plasma. After multiple infusions of Fac-

Mammalian recombinant proteins: structure, function and immunological analysis Kaufman tor VIII, approximately 15% of the patients develop antibodies that inactive Factor VIII. A possible association between certain HLA types and inhibitor formation has been suggested, but has been found to be of no predictive value. Occasionally, autoantibody inhibitors to Factor VIII may spontaneously occur in normal individuals. Factor VIII inhibitory antibodies are polyclonal in origin and consist of the IgG1 and IgG4 heavy chain subclasses. The epitopes that they recognize within Factor VIII occur predominantly within domains A2 and C2 [47] and the specificity may change over time within any single patient [48]. Initially there was optimism that monoclonal-purifled plasma-derived Factor VIII would result in less inhibitor antibodies because the protein has a higher specific activity. However, recent clinical data suggest that the frequency of antibody generation does not differ from that observed with less pure plasma-derived products. The development of novel protein pharmaceuticals has required improved methods to evaluate their potential immunogenicity. Typically, proteins are used to immunize rabbits and the antisera is collected and analyzed for antibodies that react with the native protein both before and after immunoadsorption. This procedure was used to demonstrate immunological identity between plasmaderived Factor VIII and recombinant Factor VIII produced in baby hamster kidney cells [49" "]. In contrast, immunization of rabbits with a B-domain deletion molecule resulted in unique antibodies that did not recognize wildtype recombinant Factor VII1, but did react with a peptide derived from the novel junction created by the deletion [49 ° °]. Although crossreactivity provides a valuable measure of potential immunogenicity, this procedure only allows analysis of immunodominant epitopes within a polypeptide. It cannot be used to prove immunological identity. One method that may prove useful to these ends involves the induction of neonatal tolerance in an animal to one form of the polypeptide, followed, at a later stage, by challenge with the other form of the polypeptide. This method has been used to show immunological identity between human plasma-derived and recombinant Factor VIII (E Alderman, personal communication). Altematively, transgenic mice expressing the human Factor VIII can be challenged with different preparations of the protein. The antisera developed can then be tested to identify immunological differences.

Treatment of patients with Factor VIII inhibitor antibodies The treatment of patients who have Factor VIII inhibitor antibodies presents one of the most difficult aspects of hemophilia therapy. So far, the most widely adopted approach uses activated vitamin K-dependent factor complexes that are composed of Factors Viia, IXa, Xa, and thrombin. However, severe thrombotic complications are associated with these treatments. More recently, activated Factor VII has been shown to bypass the requirement for Factor VIII [50], but very large doses are necessary to elicit hemostasis. Unfortunately, Factor VII, like Factor

IX, requires a 7-carboxylation of amino-terminal glutamic acid residues for functional activity. This process is carried out very inefficiently when Factor VII or Factor IX are overexpressed in mammalian cells. Thus, the cost of recombinant Factor VIIa will inevitably be very high. A1temative methods for bypass therapy may involve the use of tissue factor [51 ], or factor Xa with phospholipid [52 ]. Either of these approaches could be provided at a significantly reduced cost. Tissue factor can be expressed in a functional form in Escherichia coli, and the use of activated Factor X would require orders of magnitude less protein per dosage. However, the potential for thrombotic complications resulting from these bypass therapies is unknown. More direct therapy involves reduction of the inhibitor antibody in the patient's plasma. Several patients have been treated by extracorporeal immunoglobulin adsorption. Because many inhibitory antibodies appear to have common epitopes, development of specific anti-idiotypic antibodies to common epitopes may provide an alternative method to overcoming inhibitory antibodies [53 °]. Most promising is the use of high-dose Factor VIII to elicit immune suppression of the inhibitor antibody. Success has been reported in a number of clinical trials [54 o]. However, this approach is limited due to the present high cost of Factor VIII preparations. Ideally, a non-immunogenic form of Factor VIII should be developed to improve hemophilia therapy. The addition of polyethylene glycol to Factor VIII may reduce its immunogenicity, as in the case of, for example, interleukin-2. Alternatively, it may be possible to engineer carbohydrate addition sites into the Factor VIII molecule so that the immunogenic sites are covered by carbohydrates and therefore escape immune recognition. This approach is frequently used by viruses, most notably influenza virus [56].

Conclusions Our understanding of the mechanism by which Factor VIII functions in the coagulation pathway has increased tremendously over the past 10 years. The two most significant advances have been the isolation of the Factor VIII gene, approximately 7 years ago, and the ability to extract homogeneous preparations of Factor VIII from plasma and from genetically engineered mammalian cells. The availability of pure preparations of Factor VIII has provided a means of studying the biochemistry of cofactor activation and the macromolecular assembly of the Factor Xa generating complex. The identification of monoclonal antibodies against specific epitopes within Factor VIB and the use of site-directed DNA-mediated mutagenesis to generate mutant forms of Factor VIII for characterization have provided important tools to dissect the structural requirements for Factor VIII function. Our increased understanding of how Factor VIII functions in blood coagulation will have tremendous impact on the therapy available for individuals afflicted with hemophilia A. So far, recombinant DNA technology has provided homogeneous preparations of plasma-derived Factor VIII that are

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Mammalian gene studies essentially free of contaminating viruses. In the near future, recombinant-derived Factor VIII should provide an unlimited alternative source for treatment of hemophilia A. Recently, retroviral-mediated gene transfer of human Factor VIII has been achieved in tissue culture systems [57]. With further advances in gene transfer techniques in humans, it can be hoped that the use of ultrapure Factor VIII produced b y recombinant DNA technology may contribute towards an ultimate cure for hemophilia A.

10. 11.

SEVARINOKA, STORK P, VENTIMIGLIAR, MANDEL G, GOODMAN RH: Amino-terminal s e q u e n c e s of prosomatostatin direct intracellular targeting b u t n o t processing specificity. Cell 1989, 57:11-19. Anglerfish prosomatostatin is not secreted by a regulated pathwaywhen expressed in a mammalian endocrine cell line. When the 78 amino-terminal amino acids from rat prosomatostatin are inserted into anglerfish prosomatostatin, the protein is secreted by the regulated pathway. These results show that the 78 amino acids contain sufficient information to correct the targeting defect in anglerfish prosomatostatin. •

12.

Annotated references and recommended reading • • •

Of interest Of outstanding interest

1.

MANN KG, NESHEIM ME, CHURCHWR, HALEYP, KRISHNASWAMY S: Surface-dependent reactions of t h e vitamin K - d e p e n d e n t complexes. Blood 1990, 76:1-16. This is a comprehensive review of the regulation of assembly of the prothrombinase complex. The functional homology between Factor VIII and Factor V and their roles in the Factor Xa-generating complex and the prothrombinase complex are described. •

2.

3.

VEHARGA, KEYT B, EATON D, RODRIGUEZ H, O' BRIEN DPO, ROTBLAT F, OPPERMANN H, KECK R, WOOD WI, HARKINS RW, TUDDENHAM EGD, VEHAR GA, LAWNRM: Structure of h u m a n Factor VIII. Nature 1984, 312:337-342. TOOLEJJ, KNOPF JL, WOZNEY JM, SULTZMANLA, BUECKERJL, PITTMAN DD, KAUFMANRJ, BROWN E, SCHOEMAKERC, ORR EC ET AL: Molecular cloning of a cDNA encoding h u m a n antihemophilic factor. Nature 1984, 312:342-347.

KAUFMANRJ, WASLEYLC, DORNERAJ: Synthesis, processing and secretion of r e c o m b i n a n t h u m a n Factor VIII e x p r e s s e d in mammalian cells. J Biol Chem 1988, 263:6352~5362. The characterization of the biosynthesis of Factor VIII in Chinese hamster ovary cells is described. Because there are no known naturally occurring cell lines that express Factor VIII, our knowledge of Factor VIH biosynthesis is primarily based on these studies. 4.

•

PITTMANDD, KAUFMANRJ: Structure-function relationships of • Factor VIII elucidated t h r o u g h r e c o m b i n a n t DNA technology. Thromb Haemost 1989, 61:161-165. This review describes the insights that have been obtained using sitedirected DNA-mediated mutagenesis to dissect Factor VIII function. 5.

DORNERAJ, WASLEYLC, KAUFMANRJ: Protein dissociation from GRP78 and secretion are blocked by depletion o f cellular ATP Levels. Proc Natl Acad Sci USA 1990, 87:7429-7432 A demonstration that ATP is required for the release of proteins from BiP in vivo. Different proteins exhibit different ATP requirements for secretion based on their relative associations with BiP. 6.

• •

7. • o

MACHAMERCE, DOMS RW, BOLE DG, HELENIUS A, ROSE JK: Heavy chain binding protein recognizes incompletely disulfide-bonded forms of vesicular stomatitis virus G protein. J Biol Chem 1990, 265:6879-6883. These experiments demonstrate that vesicular stomatitis virus glycoprotein with intermediate disulfide bond formation binds to BiP and that, after correct disulfide bonds are formed, the protein is released from BiP.

FURmB, FUR1E BC: Molecular basis of vitamin K - d e p e n d e n t g-carboxylation. Blood 1990, 75:1753-1762.

THOMASG, THORNE BA, THOMAS L, ALLEN RG, HRUBY DE, FULLER R, THORNER J: Yeast KEX2 endopeptidase correctly cleaves a n e u r o e n d o c r i n e p r o h o r m o n e in mammalian cells. Science 1988, 241:226-230.

13. •

BIRD C, BURKE J, GLEESON PA, MCCLUSKEYJ: Expression of h u m a n immunodeficiency virus 1 (HIV-1) envelope g e n e p r o d u c t s transcribed from a heterologous promoter: kinetics of HIV-1 envelope-processing in transfected cells. J Biol Chem 1990, 265:19151-19157. This is the most detailed description of the requirements for efficient gpl60 expression and demonstrates the poor efficiency by which processing of the gpl60 occurs in heterologous cells. It also demonstrates that surface expression of gp120-41 is modulated by expression of rev. 14.

STEIN BS, ENGLEMAN EG: Intracellular processing of the g p l 6 0 HIV-1 envelope precursor. J Biol Chem 1990, 265:264(~2649.

15.

SPAETERR, SAXENAA, SCOTF PE, SONG GJ, ROBERTSWS, BRrlT WJ, GIBSON W, RASMUSSEN L, PACHL C: Sequence r e q u i r e m e n t s for proteolytic processing of glycoprotein B of h u m a n cytomegalovirus strain Towne. J Virol 1990, 64:2922-2931.

16.

FOSTERDE, SPRECHERCA, HOLLY RD, GAMBEEJE, WALKERKM, KUMARAA: Endoproteolytic processing of the dibasic cleavage site in t h e h u m a n protein C p r e c u r s o r in transfected m a m m a l i a n cells: effects of s e q u e n c e alterations o n efficiency of cleavage. Biochemistry 1990, 29:347-354. This paper describes the importance of amino acids around the propeptide cleavage site that can influence the efficiency of propeptide cleavage. It demonstrates that the substitution of a single amino acid residue, an arginine at - 4, improves the efficiency of cleavage. •

17.

MEULLmNP, B ~ D

•

C, GRANDGEORGE M, LECOCQ J-P: Increased biological activ-

A, LEPAGE P, MISCHLERF, DOTT K, HAUSS

ity of a r e c o m b i n a n t Factor IX variant carrying alanine at position + 1. Protein Engineering 1990, 3:629-633. A demonstration of h o w the efficiency of Factor IX propeptide cleavage can be improved by the substitution of a single amino acid residue, an alanine at + 1. 18. • •

WISE RJ, BARR PJ, WONG PA, KIEFER MC, BRAKEAJ, KAUFMAN RJ: Expression of a h u m a n proprotein processing enzyme: correct cleavage of t h e v o n Willebrand factor p r e c u r s o r at a paired basic amino acid site. Proc NatlAcad Sci USA 1990 (in press). The first demonstration that a cloned gene can direct the expression of a h u m a n proprotein processing enzyme. Overexpression of the processing enzyme improves the processing of heterologous proteins expressed in COS-1 monkey kidney cells. 19.

HILL-EUBANKSDE, PARKER CG, LOLLAR P: Differential proteolytic activation of Factor VIII-yon Willebrand factor c o m p l e x by thrombin. Proc Natl Acad Sci USA 1989, 86:6508~5512.

8.

prrrMAN DD, KAUFMAN RJ: Proteolytic r e q u i r e m e n t s for t h r o m b i n activation of anti-hemophillc factor (Factor VIII). Proc Natl Acad Sci USA 1988, 85:2429-2433

20.

GITSCHIERJ, KOGAN S, LEVINSON B, TUDDENHAMEGD: Mutations of Factor VIII cleavage sites in hemophilia A. Blood 1988, 72:1022-1028.

9.

WISE RJ, PITrMAN DE), HANDIN RI, KAUFMANRJ, ORKIN SH: The propeptide of y o n Willebrand factor independently mediates t h e assembly of von Willebrand multimers. Cell 1988, 52:229-236.

21.

O'BR1ENDP, TUDDENHAMEGD: Purification and characterization of Factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient w i t h severe hemophilia A. Blood 1989, 73:2117-2122.

Mammalian recombinant proteins: structure, function and immunological analysis Kaufman 22.

LOLLAR P, PARKER CG: pH-Dependent denaturation of thrombin-activated porcine Factor VIII. J Biol Chem 1990, 265:1688-1692.

23.

EATOND, RODRIGUEZ H, VEHAR GA: Proteolytic processing o f h u m a n Factor VIII. Correlation of specific cleavage by thrombin, Factor Xa, and activated p r o t e i n C w i t h activation and inactivation of Factor VIII coagulant activity. Biochemistry 1986, 25:505-512.

24. • •

WALKERFJ, SCANDELLAD, FAY PJ: Identification of t h e binding site for activated protein C o n the light chain of Factors V and VIII. J Biol Chem 1990, 265:1484-1489. This is a detailed study of the identification of an APC binding site within Factor VIII using synthetic peptides and antibodies to dissect the interaction. 25. • •

EHRLICHHJ, JASKUNAS SR, GRINNELL BW, YAN SB, BANG NU: Direct e x p r e s s i o n of r e c o m b i n a n t activated h u m a n protein C, a serine protease. J Biol Chem 1989, 264:14298-14304. Protein C requires deavage by thrombin to become activated. This pa per demonstrates that engineering a propeptide processing site into the thrombin cleavage site required for activation of h u m a n protein C results in the secretion of fully activated protein. 26.

HANSENL, BLUE YL, BARONE K, COLLEN D, LARSENGR: Punctional effects of asparagine-linked oligosaccharide o n natural and variant h u m a n tissue-type plasminogen activator. J Biol Chem 1988, 263:15713-15719.

27.

MIYAZONOK, HELDIN C: Role for carbohydrate structures in TGF-~I latency. Nature 1989, 338:158-160. Enzymatic removal in vitro of the carbohydrate structures in the TGF~1 precursor is s h o w n to produce biologically active TGF-~I from the latent complex, suggesting that carbohydrate is important in rendering TGF-I31 inactive in the complex in vivo. •

28.

29.

MOONEN P, MERMOD J, ERNST JF, HIRSCHI M, DELARMARTER JF: increased biological activity of deglycosylated recombinant g r a n u l o c y t e / m a c r o p h a g e colony-stimulating factor p r o d u c e d by yeast or animal cells. Proc Natl Acad Sci USA 1987, 84:4428-4431.

34.

EATONDL, WOOD WI, EATON D, HASS PE, HOLLINGSHEAD P, WION K, MATHERJ, LAWNRM, VEHARGA, GORMANC: Construction and characterization of an active Factor VIII variant lacking the central one-third of t h e molecule. Biochemistry 1987, 25:8342-8347.

35.

KAUFMANRJ, PITTMAN DD, WASLEYLC, FOSTER BW, AMPHLETT GW, GILES AR: Direct mutagenesis in the study of the req u i r e m e n t s for Factor VIII activity in vitro and in vivo. Thromb Haemost 1987, 58:1970a.

36. ,

WITTWER AJ, HOWARD SC: Glycosylation at Asn-184 inhibits t h e conversion of single-chain to two-chain tissuetype plasminogen activator by plasmin. Biochem 1990, 29:4175-4180. This report demonstrates that glycosylation can influence the biological activity of tPA_ It is interesting to speculate that tPA activity may be regulated by alterations in its glycosytation. 37.

38. • •

RYDErTJ, RAVlCHANDRANKG, TUnNSKY A, BODE W, HUBER R, ROITSCH C, FENTON J'q4 II: T h e structure of a c o m p l e x of r e c o m b i n a n t hirudin and h u m a n 0t-thrombin. Science 1990, 249:277-280. This work presents the structure of a complex of recombinant thrombin with himdin, providing crucial information for the development of potent anti-thrombin agents as well as increasing our understanding of setine promase function. 39.

NmHP,S C, HUTTNERWB, CARVALLOD, DEGRYSE E: Conversion of r e c o m b i n a n t hirudin to the natural form by in vitro tyrosine sulfation. J Biol Chem 1990, 265:9314-9318.

40.

NIEHRSC, HUTrNER WB: Purification and characterization of tyrosylprotein sulfotransferase. EMBO J 1990, 9:35-42.

41.

PrlTMANDE), KAUFMANRJ: Post-translational modifications important for Factor VIII function. In Proceedings of the Worm Federation of Hemophilia. 1990, Elsevier Press (in press).

42.

FOSTER PA, FULCHER CA, HOUGHTEN RA, ZlMMERMAN TS: An i m m u n o g e n i c region within amino acid residues Va1167°-Glu t684 of t h e Factor VIII light chain induces antibodies w h i c h inhibit binding of Factor VIII to y o n Wiliebrand factor. J Biol Chem 1988, 263:5230-5234.

43.

BAENZIGERJU, GREEN ED: Pituitary glycoprotein h o r m o n e oligosaccharides: structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin, and thyrotropin. Biochim Biophys Acta 1988, 947:287-306.

44.

MUTt V: Cholecystokinin-isolation, structure, and functions. In "Gastrointestinal Hormones' edited by GBJ Glass. New York: Raven Press, 1980, p p 169-221.

TAKEUCHIM, TAKASAKI S, SHIMADA M , KOBATA A: Role of sugar chains in t h e in vitro biological activity of h u m a n erythropoietin p r o d u c e d in r e c o m b i n a n t Chinese h a m s t e r ovary cells. J Biol Chem 1990, 265:12127-12130.

TAKEUCHIM, INOUE N, STRICKIANDTW, KUBOTA M, WADA M, SHIMIZUR, HOSHI S, KOZUTSUMI H, TAKASAKI S, KOBATA A: Relationship b e t w e e n sugar chain structure and biological activity of r e c o m b i n a n t h u m a n erythropoietin p r o d u c e d in Chinese h a m s t e r ovary cells. Proc Natl Acad Sci USA 1989, 86:7819-7822. A description of gtycosidase treatment of purified recombinant h u m a n EPO to evaluate the role of glycosylation in its function. The findings suggest that heavy glycosylation of EPO serves a crucial function in vivo but does not influence the interaction with the EPO receptor in vitro. 30. • •

WAStEYLC, TIMONY G, STOUDEMIRE J, DORNER AJ, CARt J, KREIGERM, KAUFMANRJ: T h e importance of n- and o-linked oligosaccharides for the biosynthesis and in vitro and in vivo biological activities of erythropoietin. Blood 1991, (in press). Studying EPO expressed in a cell line that is defective in the addition of galactose to the growing oligosaccharide chains, the authors obtain similar conclusions to [30 • •].

31. •

•

HUTrNERWB, BAEUERLEPA: Protein sulfation on tyrosinc. In Modern Cell Biology 6 edimd by Satir BH. New York: AR Liss Inc, 1988, pp 97-140.

45. • •

GRIBBENJG, DEVEREUX S, THOMAS NSB, KEIM M, JONES HM, GOLDSTONEAH, LINCHDG: D e v e l o p m e n t of antibodies to unp r o t e c t e d glycosylation sites on r e c o m b i n a n t h u m a n GMCSF. Lancet 1990, 335:434-437. This report demonstrates that proteins produced without appropriate glycosylation, i.e. in lower eukaryoms or E. coli, may elicit antibodies against the unglycosylated sims. 46.

ROSENBLUMMG, URGER BW, GUTrERMANJV, HERSH EM, DAVID GS, FRn'qCKE JM: Modification of h u m a n leukocyte interferon pharmacology w i t h a monoclonal antibody. Cancer Res 1985, 45:2421-2424.

32.

DORNERAJ, BOLE DG, KAOFMANRJ: T h e relationship of Nlinked glycosylation and heavy chain-binding p r o t e i n association w i t h t h e secretion of glycoproteins. J Cell Biol 1987, 105:2665-2674.

47.

SCANDELLAD, MATTINGLYM, DE GRAAFS, FULCHERCA: Localization of epitopes for h u m a n Factor VIII inhibitor antibodies by immunoblotting and antibody neutralization. Blood 1989, 74:1618-1626.

33.

TOOLE JJ, PrlTMAN DD, ORR EC, MURTHA P, WASLEY LC, KAUVMAN RJ: A large region ( = 9 5 kDa) of h u m a n Factor VIII is dispensible for in vitro procoagulant activity. Proc Natl Acad Sci USA 1986, 83:5939-5942.

48.

FULCHERCA, LECHNERK, MAHONEYSDE-G: I m m u n o b l o t analysis s h o w s changes in Factor VIII inhibitor chain specificity in Factor VIII inhibitor patients over time. Blood 1988, 72:1348-1356.

149

150

Mammalian gene studies 49. • •

ESMONPC, KUO HS, FOURNELMA: Characterization o f recombinant Factor VIII deletion m u t a n t using a rabbit i m m u n o genicity m o d e l system. Blood 1990, 76:1593-1600. This papers shows that a B-domain deletion mutant of Factor VIII can elicit an antibody response in a rabbit and that the resulting antibody does not crossreact with wildtype Factor ViII but does have specificity for the novel junction within the deleted molecule. 50.

HEDNERU, GLAZER S, PINGEL K, ALBERTS KA, BLOMBACKM, SCHULMAN S, JOHNSSON H: Successful u s e of recombinant Factor g i l a in patient w i t h severe haemophilia A during synovectomy. Lancet 1988, ii:1193.

51.

BOM vJJ, BERTmaARM: The contribution of Ca 2+, phospholipids, and tissue-factor apoprotein to the activation of hum a n blood-coagulation Factor X by activated Factor VII. Biochem J 1990, 215:327-336.

52.

GmESAR, MANN KG, NESHEIM ME: A combination o f Factor Xa and phosphatidylcholine-phosphatidylserine vesicles bypasses Factor VIII in vivo. B r J Haematol 1988, 69:491~i97.

LUBAHNBC, REISSNER HM: Characterization of a monoclonal anti-idiotype antibody to h u m a n anti-Factor VIII antibodies. Proc Natl Acad Sci USA 1990, 87:8232-8236. An anti-idiotype antibody to an anti-Factor VIII inhibitor antibody is shown to exist in two patients indicating the presence of inhibitory antibodies with shared idiotypes. Such anti-idiotypic antibodies will be

useful for identifying genetic factors important in the immune response to Factor VIII as well as having potential clinical use. 54. •

NltSSONLM, BERNTORP E, ZETlXRVALLO, DAHLBACKB: Noncoagulation inhibitory Factor VII1 antibodies after induction o f tolerance to Factor VIII in hemophilia A patients. Blood 1990, 75:378-383. After induction of tolerance to Factor VIII by Factor VIII treatment of hemophilia A patients with cyclophosphamide and high-dose intravenous IgG, anti-Factor VIII antibodies are still detected. However, these anti-Factor VIR antibodies differ from the original ones in specificity, lack coagulation inhibitory activity, and do not enhance the rate of elimination of Factor VIII. 55. •

KATRENV: Immunogenicity of r e c o m b i n a n t IL-2 modified by covalent attachment of polyethylene glycol. J I m m u n o l 1990, 144;20~213. PEGylated recombinant interleukin (IL)-2 is shown to be less immunogenic than unmodified IL2 in rabbits. 56.

SKEHELJJ, STEVENS DJ, DANIELS RS, DOUGLASAR, KNOSSOW M, WILSON LA, WILEYI)(2: A carbohydrate side chain o n hemagglutinins o f Hong Kong influenza viruses inhibits recognition by a monoclonal antibody. Proc Natl Acad Sci USA 1984, 81:1779-1783.

57.

ISRAELI)I, KAUFMANRJ: Retroviral-mediated transfer and amplification of a functional h u m a n Factor VIII gene. Blood 1990, 75:1074-1080.

53. •